This is to certify that the thesis entitled HISTOCHEMISTRY AND PHYSIOLOGY OF SKELETAL MUSCLE IN EXERCISE-TRAINED HAMSTERS presented by Ann Catherine Snyder has been accepted towards fulfillment of the requirements for M.A. degreein Physical Education Major professor Date Au}, '7/ W777 0-7639 A‘u'_ ‘ .' HISTOCHEMISTRY AND PHYSIOLOGY OF SKELETAL MUSCLE IN EXERCISE-TRAINED HAMSTERS By Ann Catherine Snyder A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF ARTS Department of Health, Physical Education and Recreation 1979 ABSTRACT HISTOCHEMISTRY AND PHYSIOLOGY OF SKELETAL MUSCLE IN EXERCISE-TRAINED HAMSTERS By Ann Catherine Snyder The purpose of this study was to determine the effects of aging and physical training on selected characteristics of normal hamster muscle. Twenty-seven animals were assigned to one of three groups: (a) zero-week control, (b) 12—week sedentary control, and (c) 12- week high-intensity endurance swimming. The right leg soleus (SOL) and rectus femoris (RF) muscles were examined histochemically. Muscle fiber profiles and respective fiber sizes were determined. Corresponding muscles from the left leg were examined physiologically. Muscle strength, speed, rate of relaxation and fatiguability were determined. The percentage of "fast" and "slow" fibers did not change in the central area of the SOL muscle or in the superficial area of the RF muscle. In the deep area of the RF muscle, however, the percentage of "fast" fibers was altered by both aging and training. Physiologically most characteristics were found to be affected by aging, but not altered by training. This study is dedicated to my entire family, each and everyone of them, including the "fourth daughter", for their everlasting support and encouragement which have made it all possible. ii ACKNOWLEDGMENTS I would like to express my sincere appreciation to those peOple who gave of their time and expertise throughout the course of this study. Special thanks go to Dr. William.Heusner, my advisor, whose interest in both me and my work are deeply appreciated. Thanks also go to Mr. Robert Wells who not only had the expertise to develOp the physiological characteristics data collecting system used in this study, but also displayed neverending patience in explaining its operation to me. Thanks also go to Dr. Rexford Carrow who not only Opened his laboratory to me but initiated my enthusiasm into the study of muscle. I would also like to thank Mrs. Barbara Wheaton for her time in teaching me the histo— chemical and histological techniques involved in the study of muscle. A very special thank you goes to Kevin Scribner and Sandy Herman for their neverending assistance and stimulating conversations, both of which contributed greatly in the overall preparation of this thesis. The technical assistance of Dee Jakubiak and Joie van Huss is also deeply appreciated, as is the assistance of Dr. wayne Van Russ and Bonnie Smoak. Thanks also go to Dr. Joseph Vorro for the use of his electronic digitizer which made possible the measuring of the muscle fiber areas. A final thank you goes to all those people who were not only patient but also understanding throughout the preparation of this thesis. iii TABLE LIST OF TABLES . . . . . . . . . LIST OF FIGURES . . . . . . . . CHAPTER I m PROBLEM O O O O O O O 0 Purpose of the Study . Need for the Study . . Research Hypotheses . Research Plan . . . . Limitations of the Res OF CONTENTS earch Plan II RELATED LITERATURE . . . . . . . . . Characteristics of Muscle DevelOpment of Muscle Adaptation of Muscle to Physical III METHODS AND MATERIALS . . . Experimental Animals . Treatment Groups . . . Animal Care . . . . . Sacrifice Proceedures Histochemical Procedures Tissue Analyses . . . Analysis of Data . . . IV RESULTS AND DISCUSSION . . Analysis of the Physical Analysis of Body and Muscle Weight . . . . . Histological and Histochemical Data . Physiological Characteristic Data Discussion . . . . . . O O O O O O 0 Training Performed V SUMMARY, CONCLUSIONS AND RECOMMENDATIONS . Summary . . . . . . . Conclusions . . . . . Recommendations . . . LIST OF REFERENCES . . . . . . . Page vii ix #uWNN H Ln 33 39 S4 54 54 55 55 67 67 74 75 75 76 77 87 107 115 115 117 117 119 Page APPENDICES A. Training Progrm O O I O O O O O O O O O O 0 O O O O O O O 144 B. Muscle Fiber Profile Characteristics . . . . . . . . . . . 145 10. ll. 12. 13. 14. 15. 16. LIST OF TABLES Nomenclature of Muscle Fiber Types . . . . . . . . . . Mammalian Muscle Fiber Characteristics Determined HistOChemj-cally O O O O O O O O O C O O O C O O O O 0 Fiber Type Composition of Commonly Studied Muscles . . Biochemical Evaluation of "Red" and "White" Muscle . . Muscle Fiber Characteristics Determined Biochemically Physiological Characteristics of "Slow and "Fast" MIISCIe O O O O O O C O O C C O O O O O O O C O O C O 0 Physiological Characteristics of Homogenius Muscles and Single Mbtor Units . . . . . . . . . . . . . . . . Important Muscle Fiber Characteristics . . . . . . . . Metabolically Feasible Histochemical Fiber Types . . . Fiber Type Percentages of the Central Area of the SOleus MUSCle O O O O O O O O O O O O O O O O O 0 Fiber Type Percentages of the Superficial Area of the Rectus Femoris Muscle . . . . . . . . . . . . . Fiber Type Percentages of the Deep Area of the Rectus Femoris Muscle . . . . . . . . . . . . . . . . Analysis of Variance for Overall Effects for Muscle Fiber Area . . . . . . . . . . . . . . . . . . Physiological Characteristics Obtained from Con- traction Number 1 . . . . . . . . . . . . . . . . . . Physiological Characteristics Obtained from 2-SBC contractions o o o o o o o o o o o o o o o o o 0 Physiological Characteristics Obtained from Contraction Number 12 -. . . . . . . . . . . . . . . . vi Page 16 20 22 26 29 34 72 81 81 84 86 88 9O 91 Table 17. 18. 19. 20. 21. Page Physiological Characteristics Obtained from cont rac t ion N‘lmb er 1 3 o o o o o o o o o o o o o o o o o o 93 Physiological Characteristics Obtained from contract 101). NMber 16 o o o o o o o o o o o o o o o o o o 95 Physiological Measures of Muscle Speed . . . . . . . . . . 97 Physiological Measures of Muscle Strength . . . . . . . . 99 Physiological Measures of Muscular Fatigue RESistance o o o o o o o o o o o o o o o o o o o o o o o o 101 Training Program . . . . . . . . . . . . . . . . . . . . . 144 Muscle Fiber Profile Characteristics - Fast Fibers O O O O O O O O O O O O O O O O O O O O O O O O O O 145 l U) H O t Muscle Fiber Profile Characteristics Fibers O O O O O O O O O O I O O O D O O O O O O O O O O O 146 vii Figure 1. LIST OF FIGURES Physiological Characteristic Data Collecting System Stimulation Chamber . . . . . . . . . . Electrode and Tissue Clamp Assembly of the Physiological Characteristic Data Collecting system 0 O O O O O O O I I O O O O O O O O O 0 Tissue Clamp Assembly of Physiological Characteristic Data Collecting System . . . . . Electrode Assembly of Physiological Characteristic Data Collecting System . . . . . Electrode Plating Assembly for Physiological Characteristic Data Collecting System . . . . . Stimulator waveforms of Physiological Characteristic Data Collecting System . . . . . Soleus and Rectus Femoris Muscle Areas Examined viii Page 57 58 59 61 62 64 71 CHAPTER I THE PROBLEM Histochemically, biochemically and physiologically muscle fibers have been categorized into three basic types. Different schemes have been used to describe these types; however, the nomenclature of slowh twitch—oxidative (SO), fast-twitch-oxidative-glycolytic (FOG), and fast-twitcheglycolytic (PC) as devised by Peter 35 31. (276) has become widely used. The SO fiber type is slow contracting and has high oxidative capabilities. The FOG and FG fibers are both fast contract- ing. The FOG fibers have high oxidative and glycolytic capacities, while the PG fibers have only high glycolytic capacity. The properties of muscle fibers have been found to change with deveIOpment (52,57,82,83,90,91,130,177,178,l84,204,205,229,234,242,316), immobilization (37,38,123,l45,239,240), cross-innervation (S6,S9,61,174, 233,289,292,293), denervation (ll3,176,216,283), and electrical stimulation (2,21,33,53,63,94,149,l75,181,227,270,277,278). Similar changes have been observed in the muscles of physically trained humans (3,27,76,92,93,95,l60,161,216,280,321) and animals (13,41,114,115,119, 121,142,143,195,244,295,315,317). The degree of adaptation with phys- ical training appears to vary with different species and exercise regimens (171,194,19S,295,306,317). In particular, endurance-type training programs have been found to increase the aerobic capabilities of muscle fibers (3,27,92,93,95,128,l60,216,321). A change in the l 2 percentage of fiber types, toward more oxidative fibers, also has been observed with this type of training (76,92,93,l60). Purpose of the Study The overall purpose of this investigation was to determine the effects of growth and development and physical training on selected physiological and histochemical characteristics of normal hamster soleus (SOL) and rectus femoris (RF) muscles. The animals were trained with a high-intensity endurance swimming program.for 12 weeks. Muscle fibers from the right hindlimb were examined histochemically for muscle fiber type and size. Physiological characteristics, including data on both twitch and tetanic contractions, were determined for comparable muscles from the left hindlimb. Zero-week (7 weeks of age) and lZ-week (19 weeks of age) sedentary animals served as pre- and post-training controls. Need for the Study This study was designed to fill the void which exists in the scientific literature concerning the histochemical and physiological characteristics of hamster muscle. Because descriptive work utilizing hamsters with muscular dystrOphy (4,19,39,187,213,250,241,253,254,259, 260,327) is being carried out, comparative data on normal hamster muscle is necessary. Research also is being done on the effects of physical activity in dystrophic hamsters (39), and thus training data in unaffected animals is prerequisite to an understanding of the physiological adaptations that may occur under these conditions. In addition, the relationship between the physiological characteristics and the muscle fiber metabolic characteristics have rarely been studied 3 in hamster muscle (154). The effects of physical training on these parameters have never been determined. Research Hypotheses The main hypothesis of this study was that normal hamster muscle will adapt to a physical training program of swimming. Within this hypothesis are three subhypotheses: l. Skeletal muscle fibers of hamsters will not change in size but will develOp more aerobic metabolic capacity through an endurance swimming regimen. 2. Hamster muscle will become slower contracting and more resistant to fatigue through an endurance swimming regimen. 3. Physiological and metabolic characteristics of hamster muscle are related in both sedentary and trained animals. Research Plan Twenty-seven normal Syrian hamsters were used in this study. The animals were assigned randomly to one of the following groups: (a) zero-week control (n a 8), (b) lZ-week sedentary control (n - 11), and (c) lZ-week high-intensity endurance swimming (n = 8). The left and right SOL and RF muscles were evaluated. Muscle fiber profiles and respective fiber sizes were determined using 30 adjacent fibers from the center of the SOL muscle and from the superficial and deep areas of the right RF muscle. The histochemical profiles were determined using the following stains: (a) adenosine triphosphatase at a pH of 9.4 (ATPase) as an indicator of contractile elements, (b) reduced nicotinamide adenine dinucleotide-diaphorase (NADH) for the demonstration of oxidative capacity, (c) a-glycero- phosphate dehydrogenase (aGPD), as an indicator of aerobic glycolysis, 4 (d) Sudan black B (SUD) for lipid storage, and (e) periodic acid-Schiff reaction (PAS) for glycogen storage. Corresponding muscles from the left leg were used to evaluate selected physiological characteristics. Muscle strength, speed, rate of relaxation, and fatiguability were determined for both the SOL and RF muscles. The strength measures were corrected for body and muscle weight. Limitations of the Research Plan The results of this study can be applied only to normal hamr sters. Evaluation of the effects of the training program is limited by a lack of ability to determine the intensity of muscular activity when laboratory animals are subjected to a regimen of forced swimming. The limited number of histochemical and physiological character- istics that could be determined do not provide a complete analysis of the metabolic adaptation of hamster muscle to physical training. Due to the complexity of the sacrifice, some muscles were neces- sarily exposed to air for periods of up to four minutes between the time of removal of the tissue and the time of rapid freez- ing. Due to the time required to obtain the physiological data on each muscle, the left SOL muscle was placed in Ringer's solution for approximately 15 minutes while the RF muscle was being tested. CHAPTER II RELATED LITERATURE The purpose of this investigation was to determine the effects of physical training on the physiological and histochemical characteristics of normal hamster muscle. The literature related to this investigation will be reviewed in three main sections: (a) characteristics of muscle, (b) development of muscle, and (c) adaptation of muscle to physical training. Characteristics of Muscle. Muscle has been studied histochemically, biochemically, and physiologically. Thus, this section is subdivided to permit a separate examination of the literature resulting from each of these methods of investigation. Characteristics of Muscle Determined Histochemically Fiber types and sizes have been determined from the histochemical study of muscle. Therefore, this segment of the discussion will cover both entities. Types of Muscle Fibers The fact that there are different types of muscles has been known for many years. Ranvier (286) was cited by Kronecker and Stirling (226) in 1880 as one of the first to observe that there are "red" and "white" 6 muscles. Throughout the last 100 years, a great quantity of research has taken place involving muscles and muscle fibers. Histochemical techniques have revealed distinct metabolic characteristics for each of the fiber types (125). Furthermore, longitudinal sections of muscle fibers have shown that fibers are uniform throughout their entire length (106,107). This observation makes histochemistry appear to be an apprOpriate method for determining muscle fiber type. Confusion has arisen due to inconsistencies within the literature concerning the nomenclature used to describe the various muscle fiber types. Table 1 contains a summary of the terms used to type muscle fibers. Not only has the variation in nomenclature caused researchers difficulty, but the total number of fiber types has been widely disputed. Engel (134,135) and others (31,34,36,46,92,127,129,156,162,l63,164,l65, 174,188,210,211,215,225,228,234,27l,288,289,300,304,312) contend that there are only two fiber types, while Brooke and coworkers (48,49) and many others (3,5,6,7,8,9,13,l4,lS,l6,18,23,25,28,37,38,40,55,98,ll4,ll6, 118,121,122,l37,138,140,150,151,l60,l66,l67,178,180,183,208,212,213,214, 221,222,223,224,227,231,232,239,244,249,255,264,267,273,274,275,276,280, 282,283,29l,295,308,311,313,322,330) have shown the existence of three fiber types. Observations by a few investigators (105,119,182,209,218, 224,257,258,290,296,307) have revealed as many as eight distinct types. Since most researchers have agreed that three basic fiber types exist, that categorization will be adOpted for the purpose of this review whenever practical. Table 2 contains a summary of the character- istics of the mammalian muscle fiber types using histochemical tech- niques. The nomenclature introduced by Peter et al. (276) has been used. SO III RED INT INT SO C I RED SLOW TWITCH RED BR 8 F,G&H I I INT, MOD SLOW TWITCH RED INT Nomenclature of Muscle Fiber Types FOG IIA II FO B II RED FAST TWITCH RED aR D&E III INT RED INT RED TYPE I OXIDATIVE 7 Table 1 II WHITE WHITE TYPE II GLYCOLYTIC FG IIB WHITE II F A II WHITE FAST TWITCH WH 0W A,B&C II II WHITE FAST TWITCH WHITE 5,6,121,124,137, 151,166,167,239, 276,280,295 3,48,49,98,138,231, 232,282 31,46,127,134,13S, 156,188,210,211, 215,228,271,288, 289,300 291 36,225,234,304 13,14,16,23,25,28, 37,114,116,118,140, 150,255,264,267,283 249,313,322 38 55,227,330 222,223,224 18,40,122,160,172, 274,275 7,8,9 174 290 213,214 208 119,258 34,98,129,182,163, 164,165,312 212 221 Dm ST HIGH OXIDATIVE SLOW (HS) S SLOW TWITCH INT SLOW TWITCH RED B SLOW TWITCH MOD. OXIDATIVE INT SLOW TWITCH OXIDATIVE 8 Table 1 (continued) Ld FTH HIGH OXIDATIVE FAST (HF) FR FAST TWITCH RED FAST TWITCH INT C FAST TWITCH HIGH OXIDATIVE RED FAST TWITCH OXIDATIVE GLYCOLYTIC L1 FT LOW OXIDATIVE FAST (LF) FF FAST TWITCH WHITE FAST TWITCH WHITE A FAST TWITCH LOW OXIDATIVE WHITE FAST TWITCH GLYCOLYTIC 257,258 180,308 244 183 25,273 25 311 15 121 som.ss~ was .wAm .o-.m-.mmA.smA uoefiao .omuom .uao .NNA.8AA.em.Am .umm .amasm .mmsegz a a A and mmmuommdmua mmA.smA case: a A amwouonuomo: «s.on poo H A a mammaom .mmagz .s~.-.o~.ao.ae .wam mmeAso .uoumaam .q¢.wm.Am.m~.m .umo .umm .amaam a an mA qumeHw ommme< moueuuomoe muomfiaam we won om maAMOfiaosooumwm voafiauouon mouumfiuouumumfio Hanan vomaz cmfiamaamz N canoe 10 AA use A A A AAA «om mmaoz A 91H H mnA Aom.eNN.moN AAAAAA .wAA maeAsu .osA.mmA.smA.AoA .uau .Amasm .AAA A A A AAA oumnmmonm «AN AAAAAA A A A umnAoAAUAAo ommaowouvhnov NAN use A A A muAAAmoAAomoAAA AAA NNA.NAN.ANA.8N smAAau .amasm .umo A A A oAuAAouAAu «AN «man: A A A AAus Aom.mAN.AAA.sAA NAA maaAsu .AAA .AAA A A A AAos ANN.ANN.NNN.NNN .AAA.AAA.8AA.AA amaze .umo A A A AAos NAN Ame AIA AIA AuA AAA wom.sNN ounce .AAA.AAA.AAA.AA .AAA «AAAAA .880 A A A AAA nnN AAA A A A AAA AAAAAA AAN.NAN.ANN.ANN .umo .meAuqu .mAA>oA .eAN.mAN.woN.A .mmaoz .Nmumamm .umA A HIA A AAA mooaouomom muoofinam cm com ow Awwsafiuaoov N manna 11 AAN.AAN.AAN.AAN .AAA.AAA.AAA.AAA AAA .mAAoA .NA.AA.AA.AA.AA .Amumamm .umo .Amasm A A-A A AAoA .oAA.AAA.AAA.sAA .omaoz .umo .oneoz .AoA.NA.oA.AA.A .Amasm .AAAAAA .AAA A AuA A AAA As amen: A A A m>AAmAAxo omN.AAN.ANA seas: .AAA A A AuA m>AumeAxo ommcowouphnov #0 N 9352 mcoc A OdoAA 333356 on use a A A ommuoumm ANN.¢AA Ana .Amasm A A A «Amuoumm , AAAAAAAAAA muuohAsA AA AAA oA Ammaaaucoov N manna 12 zufimcouew mafiaamum oumuovoa a Adamoavad Ho auwmaousa maweamum Amwn a mouoowwqu n A augm:MAAH weaswmum 30H m moumowvca Am am: 325% 3.8 .AAA omN.AoN.AoN AAAAAA .AAA .owaoz A AuA A AAAoAAoAz AAA AAA A A A see Aom AAAoA A A A AAA NAN Ame A A A AAA AAA.AAA.AA AAA .Amasm A A A 0 AAA AAA EmHHonmumz omN.ANA 8AA .Amaam A A A AAAAA AoAAAAAAAAA omN Ann 9 H A sowOUAHU How ommuwxd .AAN.ANN.AAA.AAA amass .AAAAAA .AAA A A AIA «sounquAu ANN uAA A A A AAAz woocmummom muuownsm um com om amenaAAAOOV N manna 13 The SO fiber is characterized by intermediate to high oxidative capability (as determined by succinic dehydrogenase--SDH, NADH, and malic dehydrogenase--MDH), low aerobic glycolytic activity (aGPD) and a slow contraction time (ATPase). Glycogenolytic (phosphorylase—-PPL) activity and lactate formation (lactate dehydrogenase—-LDH) are both low. Lipid storage as determined by SUD and 011 Red 0 is high in these fibers, while glycogen storage (PAS) has been found to be low. The FOG muscle fiber has extensive metabolic capabilities as indicated by its intermediate to high oxidative, glycogenolytic and aerobic glycolytic activities. The FOG muscle fiber also has a fast time of contraction. Glycogen storage is high in this fiber, and lactate formation is intermediate to high. Lipid storage is inter- mediate. The FG muscle fiber also is fast contracting and high in glycogen- olytic activity, lactate formation and aerobic glycolytic activity. However, unlike the FOG fiber, the PG fiber is very low in oxidative enzyme activity as well as in lipid storage. Glycogen storage in the FG muscle fiber is high. The mitochondrial content of the FOG fiber is high (273) which has been distinguished by the abundance of coarse diformazan granules seen with an NADH stain (7,25). The SO fiber has an intermediate content of mitochondria and thus uniformly dispersed diformazan granules. The PG fiber has low mitochondrial concentration and few diformazan granules. Although the information given in Table 2 appears to be fairly consistent, many stains reappear in order to accommodate the varying staining patterns observed by different investigators. There appear to be several reasons why all muscle fibers can not unquestionably be categorized within one of the three main groups: (a) muscle fibers 14 from different species appear to be different, (b) muscle fibers from different locations within the Same species have been found to be different, and (c) muscle fibers may actually be a continuum, Guth and Yellin (173) and Yellin and Guth (330) concluded that muscle fiber types between species are different. Their work dealt with. rats, cats, mice, guinea pigs, and rabbits. All muscles examined revealed somewhat similar fiber types; however, the intensity of the histochemical staining was different between the muscles. Engel and Irwin (133) and Sarnat gt_§l, (298) found the following three types of frog muscle fibers: (3) a type of fiber that is high in oxidative capacity, glycogenolytic activity, and glycogen storage, (b) a second fiber type that is intermediate in the aforementioned categories, and (c) a third fiber type that is low in all three. These amphibious fiber types are all different from those reflected in Table 2. Brooke and Kaiser (50) found that in the human biceps brachii the TYPE IIA fiber is an intermediate muscle fiber, while the TYPE IIA fiber of the rat gastrocnemius muscle is a red fiber when the same classifying systems are used to determine the muscle fiber types. Peter (273), aware of this discrepancy in the oxidative capacity between human and animal muscle fibers, suggested a terminology of fast-twitch white, fast-twitch intermediate and slowbtwitch red for human muscle while using fast-twitch white, fast-twitch red and slowbtwitch inter- mediate for animal muscle. In studies involving rats and cats, the intensity of staining was different between two muscles of the same animal even though the fibers were classified as the same type (74,99,227,263). These studies used a variety of histochemical stains to compare the soleus with the 15 gastrocnemius (74,99), the rectus femoris (263), and the anterior tibial muscle (227). A number of investigators (ll4,l6l,235,273,296) have prOposed the existence of a continuous spectrum of muscle fibers. This point is supported by the studies within a single muscle of the same species in which some of the muscle fibers could not be classified as belonging to one of the three basic fiber types (105,119,182,209,218,224,228,257,258, 296,307). Some of these investigators (105,ll9,182,209,218,257,258,307) distinguished four fiber types, while Khan (224) found seven and Romanul (2 90) eight. Muscle fiber types have been found to be distributed through a muscle (13,29,129,137,235,282,307). In many muscles the superficial area is composed of predominately FG fibers, while the deeper areas are primarily SO and FOG fibers (13,29,139,307). This observation applies to both human (129) and animal (13,29,137,235,282,307) subjects. Muscles from within and between species are composed of different percentages of fiber types. Table 3 summarizes the various percentages of muscle fiber types obtained by several investigators. Even though variations have been found between different muscles, Karpati and Engel (217) found a nonsignificant difference when compar- ing the proportions of fiber types in right and left leg muscles of guinea pigs. The muscles examined were the gastrocnemius, plantaris and soleus. The largest difference was 4% which was found in the small head of the gastrocnemius. Sizes of Muscle Fibers The size of individual muscle fibers has been investigated by numerous researchers (47,48,69,72,116,210,211,212,214,227,229,24l,269, 16 Table 3 Fiber Type Composition of Commonly Studied Muscles Muscle SO FOG FG References Soleus Human 64% 17% 19% 124 Human 89 7 4 6 Cat 100 5 Guinea Pig 100 5,25,273,276,289 Bushbaby 87 13 5 Slow Loris 72 7 21 5 Rabbit 96 276 Mouse 50 50 8 Biceps Brachii Human 25 32 43 55 Lateral Triceps Human 2 48 50 SS Gastrocnemius Human 52 16 31 127 Human 82 8 10 55 Medial Gastrocnemius Guinea Pig 12 50 38 25,273 Guinea Pig 22 54 24 5 Rat 4 58 38 5 Cat 25 61 14 5 Lateral Gastrocnemius Guinea Pig 12 56 32 5 Rat 5 58 37 5 Cat 18 66 16 5 Anterior Tibial Human 46 35 19 6 Rectus Femoris Guinea Pig 6 43 51 5 Rat 4 54 42 5 Cat 22 17 61 5 Bushbaby 8 48 44 5 Slow Loris 50 12 38 5 Adductor Muscles Rat 50 25 25 28 Vastus Lateralis Human 46 20 34 124 Vastus Intermed ius Human 52 15 33 124 17 Table 3 (continued) SO FOG FG References Red Vastus Guinea Pig 4% 78% 182 25,273,276 Flexor Hallicus Longus Guinea Pig 17 10 73 25,273 Flexor Digitorum Longus Guinea Pig ll 35 54 25,273 Guinea Pig 14 56 30 5 Rat 8 37 55 5 Cat 7 61 32 5 Bushbaby 7 38 54 5 Extensor Digitorum Longus Guinea Pig 6 57 37 5 Rat 3 38 59 5 Cat 14 55 31 S Bushbaby 2 45 53 5 Plantaris Guinea Pig 6 73 23 5 Rat 6 41 53 5 Cat 26 46 28 5 Bushbaby 19 56 30 5 l8 279,288,299,323). The studies on animals have shown that the three fiber types have different sizes (69,72,116,212,214,227,229,24l,299,323). JOhnson and Pearse (214) reported only that SO and FOG fibers are significantly smaller than FG fibers. Kugelburg (229) found the FG fibers to be largest, SO fibers intermediate, and FOG fibers the small— est. Most authors, however, have found the FG fibers to be largest, FOG fibers intermediate and the SO fibers the smallest (69,72,116,212,227, 241,299,323). The results obtained from human data are not as clear as those from animals. Reniers g£_al, (288) and Polgar Efinfll: (269) found FOG and FG (TYPE II) fibers to be larger than SO (TYPE I) fibers, while Jennekens g£_§l, (211) found FOG and FG (TYPE II) fibers to be inconsistently different from SO (TYPE I) fibers. Polgar SE 21. (279) also found a nonsignificant difference between the sizes of SO, FOG and FG fibers in the soleus. Jennekens 35 al, (210) reported that in the biceps brachii and deltoid muscles FOG and FG (TYPE II) fibers were the largest, whereas in the rectus femoris and gastrocnemius muscles SO (TYPE I) fibers were the largest. Brooke and Engel (47) found FOG and FG (TYPE II) fibers to be larger than SO (TYPE I) fibers in males, but the converse was true for females. Brooke and Kaiser (48) in concurrence with this observation found the SO (TYPE I) fibers for males and females to be approximately equal in size, while the females' FOG and FG (TYPE II) fibers were much smaller than the males'. Various factors such as the age (47), weight (47), and physical build (279) of the subject appear to have no significant effect on muscle fiber size once the growth period has ceased. However, Aherne g£_§l, (1) found a trend of increasing fiber size with increasing body size. 19 Goldspink (152), looking at mice, and Vincelette and Jasmin (323), studying rats, found fibers of the same size to be approximately equal in enzymatic activity; whereas Hammarberg (182), in identifying four types of muscle fibers in cats, found a considerable overlap of fiber sizes between the types. In pig longissimus dorsi and trapezius muscles, Moody and Cassens (255) found little difference in muscle fiber sizes from one muscle to another. The relationship in animal muscle fibers is linear between fiber diameter an myofibrillar number (152). A negative relationship exists between myofibrillar free sarc0plasm and muscle fiber size (152). Dulhunty st 21, (111) found a significant correlation between the total capacity of the surface membrane and fiber size as well as between the total conductance of the surface membrane and fiber size. Characteristics of Muscle Determined Biochemically Until recently the biochemical evaluation of single muscle fibers was not possible. Therefore, most biochemists have used muscles which were homogeneous or fairly homogeneous as to fiber types. Tables 4 and 5 summarize the data on muscle which has been collected biochemically. Table 4 consists of the data obtained on muscles which were described as being "red" or "white". Table 5 consists of the data obtained on muscles which were characterized as containing predominantly SO, FOG or PC fiber types. The biochemical analyses generally substatiate the histochemical data. Muscle containing predominantly SO fibers is highly oxidative (MDH, SDH, isocitrate dehydrogenase--IDH, cytochrome oxidase—-CO), slow contracting (actomyosin ATPase, myosin ATPase, KI, Na+), low in glycolytic enzymes (phosphofructokinase-PFK, pyruvate kinase-PK) and Biochemical Evaluation of "Red" and "White" Muscle 20 Table 4 "RED" "WHITE" Subjects References Myosin ATPase Low High Hamster, Guinea 25, 154 Pig Ca2+ ATPase Low High Rabbit 20 ITPase Low' High Rabbit 20 EDTAPATPase Low High Rabbit 20 Actomyosin Low High Rat, Guinea Pig 18, 25 AMEATPase Low High Rabbit 20 Total Creatine Low High Hamster 154 Calcium Uptake Low High Cat, Rat 43 Calcium Oxalate Low High Cat, Rat 43 Sarcoplasmic Proteins Low High Rabbit 20 Stroma Proteins High Low Rabbit 20 Total PPL Low High Human, Rat, 26, 36, 188 Monkey, Rabbit Active PPL Low High Rat, Monkey 36 Glycogen Low High Rat 28 Lactate Production Low High Rat 28 Specific Activity High Low Rat 28 of Lactate Total LDH Low High Rat, Rabbit 26, 160 Oxidative Capacity High Low Rat 13 Triosephosphate Low High Rabbit 26 Dehydrogenase aGPD Low' High Rabbit 26 Hexosediphosphate Low High Rabbit 26 Hexokinase High Low' Rat, Rabbit 26, 96 Citrate Synthase High Low Rabbit 26 21 Table 4 (continued) "WHITE" "RED" Subjects References Alkaline Proteinase High Low Hamster 253 Arylamidase High Low Hamster 253 Cathepsin Bl Low High Hamster 253 Glycerol-B-Phosphate Low High Rat 96 Dehydrogenase Carnitine High Low Rat 220 Palmityltransferase Carnitine High Low Rat 220 Acetyltransferase Total Carnitine Low High Rat 220 22 Table 5 Muscle Fiber Characteristics Determined Biochemically SO FOG FG Subjects References Myosin ATPase Low High High Guinea Pig 75, 120 Actomyosin ATPase Low High High Guinea Pig, 75, 120, Human 138 Arylsulfatase A High Int Low Guinea Pig 275 B-Acetyl High Int Low Guinea Fig 275 Glucosamidase B-Galactosidase High Int Low Guinea Pig 275 Acid Cathepsin High Int Low Guinea Pig 275 Acid Phosphatase High Int Low Guinea Pig 275 Total Lipids High Low Low Guinea Pig, 144 Rabbit Phospholipids Int High Low Guinea Pig 144 Cholesterol High Low Low Guinea Pig 144 Content Rabbit K+ Low High High Human 138 Na+ Low High High Human 138 Glycogen Low High Int Pig, Guinea Pig, 8, 75, 150 Mice, Bovine PPL Low Int High Guinea Pig, Rat 75, 99 Hexokinase High Int Low' Guinea Pig 75 PFK Low High High Human 138 aGPD Low High High Guinea Pig 75 NAD-aGPD Low Int High Rat 99 Aldolase Low' Int High Rat 99 Pyruvate Kinase LOW’ Int High Rat 99 M-Type LDH Low Int High Guinea Pig, Rat 99, 274 H-Type LDH High Int Low Rat 99 23 Table 5 (continued) SO FOG FG Subjects References Glucose-6-Phosphate High Int Low Rat 99 Dehydrogenase Glutamic-Oxalacetic High Int Low' Rat 99 Transaminase Creatine Low Int High Guinea Pig 75 Phosphokinase SDH High Int Low Human 138 SDH Int High Low Guinea Fig 75 MDH High Int Low Rat 99 IDH High Int Low Rat 99 Cytochrome C Int High Low Guinea Pig 75 Cytochrome A Int High Low Guinea Pig 75 Cytochrome Oxidase High Int Low Rat 99 24 low in glycogenolytic enzymes (PPL). The $0 muscle also is low in the formation of lactate (m-type LDH) and in aGPD which is needed for the regeneration of NAD+ in glycolysis. The FOG muscle is moderately to highly oxidative, moderately to highly glycolytic and fast contracting. The glycogenolytic and lactate formation enzymes for this muscle are moderately active, while the enzymes of the a-glycerophosphate shuttle are moderately to highly active. The PG muscle is high in glycolytic and glycogenolytic activity, lactate formation, and a—glycerophosphate shuttle activity. Low oxida— tive enzyme levels and fast contraction time also are apparent in Table 5. In agreement with the histochemical study by Karpati and Engel (217), Baldwin and Tipton (15) found high and very high correlations between biochemical analyses of the right and left tibialis anterior muscles in humans. The lowest correlation was found for lactic acid (r = 0.86), while total and free-carnitine each had a correlation coefficient of 0.99. Glycogen (r'- 0.91), acyl-carnitine (r’a 0.91) and phosphocreatine (r a 0.92) also were analyzed. Thomson §£_§1, (319) found no sex differences when studying the vastus lateralis muscle of humans. Their investigation included SDH, LDH, glycogen, and phosphagen concentration for either ATP or creatine phosphate. Recently biochemical (138,139,193) and physiological (69,70,7l,72, 73,74,75) data have been determined on individual muscle fibers and single motor units. The biochemical data on individual fibers corres— pond quite closely to that on whole muscle. The contractilability (ATPase) of the FOG and FG fibers was reported to be approximately 2.5 25 times higher than that of the SO fibers (138). Oxidative capacity (SDH) was found to be highest in SO fibers, intermediate in FOG fibers and lowest in FG fibers (138). The quantity of PFK was significantly higher in the PG and FOG fibers than in the SO fibers, while the reverse was true with the concentration of triglycerides (138). The amounts of glycogen in all three muscle fiber types were found to be similar (138). Characteristics of Muscle Determined Physiologically This section will deal with the physiological characteristics of muscles. The interrelationships of these characteristics and their dependence upon temperature also will be examined. Physiological Characteristics of Muscle Parallel to the finding of different muscle fiber types histo- chemically and biochemically was the observance of a difference in the physiological characteristics of muscle (226). Data are available on characteristics such as contraction time, tension produced, twitch/ tetanus ratio and rate of fatigue. As was found with some muscles studied biochemically, many researchers categorized the muscles only as "fast" or "slow". The data collected in this fashion are presented in Table 6. The distinguishing characteristics of a "slow" muscle are: slow contraction time, high mean tension, low twitch/tetanus ratio and slow rate of fatigue. A "fast" muscle exhibits the Opposite characteristics: fast contraction time, low mean tension, high twitch/tetanus ratio and rapid fatigue. With the advent of muscle histochemistry and the ability to differentiate the fibers into different types, muscle physiologists began to study muscles with a high percentage of one fiber type. In 26 Table 6 Physiological Characteristics of "Slow" and "Fast" Muscles Slow Muscles Fast Muscles Subjects References Mean Isometric High Low Rat 302 Tension Rate of Tension Low High Cat, Guinea 45,85,297, Development Pig, Ra bb it , 302 Twitch/Tetanus Low High Cat 62,256,297 Conduction Low High Cat 58,59,62, Velocity 256 Total Action Low High Cat 58 Potential Twitch Contraction Low High Cat, 58,62,226, Time Rabb it 2 56 Rate of Fatigue Low High Hamster 154 Maximum Velocity Low High Hamster 154 of Shortening Electrical Low High Human 112 Threshold 27 conjunction with other technical advancements, the study of single motor units became possible. A motor unit has been defined as being "an alpha-motoneuron and all of the skeletal muscle fibers that are functionally innervated by that neuron (126)." One muscle fiber usually is innervated by only one neuron (31,247); however, multiple innervations have been shown (247). Burke and coworkers (69,70,7l,72,73,74,75) have classified motor units into three types: (a) fast twitch fatigue sensitive (FF), (b) fast twitch fatigue resistant (FR), and (c) slow twitch fatigue resistant (S). As the appellations suggest the variables of contraction time and fatiguability were used in determining the motor unit types. More specifically, the variables used were a fatigue index and the presence or absence of "sag". The fatigue index is the ratio of maximum tension produced before and after two minutes of stimulation at a rate of 40/sec (70,73). This ratio is, therefore, a measure of the fatiguability of the motor unit. The phenomena of "sag" is characterized by an early tension maximum in the course of an unfused tetanus with a subsequent slight decline in tension to a lower plateau (70,73). This measure is an indication of contraction time since a fast fiber will reach an early tension maximum and then decline while a slow fiber will take a much longer time to reach its maximum tension. In using these two parameters to determine physiological motor unit types, Burke and coworkers (72,73) were able to classify all but 2-32 of the units they studied into S, FR, or FF types. Kugelberg (228), also studying single motor units, found that not all units could be classified into one of the three types. Although no percentage of unclassifiable units was given, only a few examples were noted and most units appeared to be one of the three types. 28 Table 7 sumarizes the data collected with respect to contractile characteristics. From this table it can be seen that the 8 type motor unit is characterized as having a high fatigue index, no "sag" present, slow contraction time, slow axonal conduction time and slow one-half relaxation time. The FR motor unit has a high fatigue index, "sag" present, intermediate axonal conduction time, fast contraction time and fast one-half relaxation time. The FF motor unit has a low fatigue index, "sag" present, fast contraction time, fast axonal conduction time and fast one-half relaxation time. Burke (64) originally found the twitch/tetanus ratio to be small in S motor units and large in FR and FF motor units. He later found this ratio to be small in S units, intermediate in FR units and large in FF motor units (70). Close (84), on the other hand, found the twitch/tetanus ratio to be virtually the same for all three types. Aside from the twitch/tetanus ratio just discussed, no overlapping of the physiological characteristics between the three types of motor units was observed (72). Muscle fibers of the same motor unit type in the same muscle have identical histochemical profiles (42,69,70,72,l34). In comparing the physiological properties of motor units in the soleus and gastrocnemius muscles, Burke and coworkers (64,72,74) did find differences within the S type units. As compared to those in the gastrocnemius, the S units of the soleus muscle have a slower contrac- tion time, slower conducting axons, no post tetanic potentiation of twitch tension, and a greater maximum tetanus tension output. Olson and Swett (266), however, concluded that one of the motor units found in the medial gastrocnemius muscle closely resembles the S motor units found in the soleus muscle. 29 Table 7 Physiological Characteristics of Homogenius Muscles and Single Motor Units S FR FF Subjects References Twitch Slow Fast Fast Guinea Pig, 25,55,64,66, Cat, Human, 67,69,70,72, Rat 75,84,228, 267,273 Peak Isometric Low Int High Cat, Rat 64,228 Twitch Tension Maximum. LOW’ Int High Cat 53,69,70,75 Tetanic Tension Maximum Isometric Low High Low Rat 84 Tetanic Tension Fatigue Index > 0.75 > 0.75 < 0.25 Cat 53,69,70,73, 75 'Sag' Absent Present Present Cat 53,64,69,70, 73,75 Twitch/Tetanus --- Virtually the same - Rat 84 Twitch/Tetanus Low Int High Cat 70 Tetanus/Twitch High Low Low' Cat 73 Mbtoneuron High Low Low Cat 64,65,66 Input Resistance Axonal Conduction Slow Faster Fastest Cat 64,66,70,75 Velocity EPSP Long Inter- Short Cat 66 Dura- mediate Duration tion Duration Post Spike Long Short Short Cat 64 Hyperpolariza- tions OneAHalf . Slow Fast Fast Rat 84 Relaxation Time Tonic Firing 100% 70% 10% Cat 66 30 Harris and Wilson (186) estimated there are 191:26 motor units in the tibialis anterior muscle of the mouse. This number is slightly higher than the 90 motor units Olson and Swett (265) found in the flexor digitorum longus muscle of a cat. Close (84) found an average of 40 motor units in the extensor digitorum longus muscle and 30 motor units in the soleus muscle of the rat. Of the 30 motor units in the soleus muscle, 3 were thought to be of the FR type with the remaining 27 being of the S type. Burke and Tsairis (71) and Wuerker 32 al, (320) indicated that fast motor units are larger than slow motor units. In the cat medial gastrocnemius muscle, Burke and Tsairis (71) estimated each FF motor unit to contain 550-750 muscle fibers, each FR motor unit to contain 400-550 muscle fibers, and each 8 motor unit to contain at least 200 fibers. Burke and Tsairis (71) also estimated that this muscle contains approximately 170,000 muscle fibers. These data are considerably higher than the 118 average muscle fibers per motor unit that Brand- stater and Lambert (42) found in the tibialis anterior muscle of the rat. Muscle fibers of individual motor units have been found to be randomly distributed throughout the muscle (42,228). Brandstater and Lambert (42) found that 762 of the muscle fibers from one motor unit were not in contact with a muscle fiber from the same motor unit. Kugelberg (228) has stated that muscle fibers of a single motor unit may cover an area of 25-752 of the total muscle, but Brandstater and Lambert (42) found this area to be only about 122. 31 Interrelationships of Physiological Characteristics of Muscle Many investigations have been performed to determine the relation- ships of physiological characteristics to each other as well as to other parameters. Maximum tetanic tension is significantly related to the rate of tension develOpment (35,318), and twitch contraction time is significantly related to the maximum rate of rise of twitch tension. However, contraction time is not correlated with the age of the subject (55,245), the twitch/tetanus ratio (84,256), or the size of the motor unit contracting (84). One—half relaxation time is significantly correlated with rate of tension development (318), rate of relaxation (318), muscle weight (318), and cross-sectional area (318) but is not correlated with the age of the subject (245). The sex of the subject does not appear to affect any of the physiological characteristics of muscle (55,245,318). In accord with the histochemical studies (50,173,273,330) which determined that muscles of the same type may be different between species, Close (82) found that both "fast" and "slow" muscles of mice contract faster than do the same muscles in rats. Although contraction time has been used historically to differenti- ate muscle types, recent questions have been posed as to the validity of this technique. Even though at least three different types of muscle fibers are known to exist, only two speeds of contraction have been identified (87). Therefore, when contraction time is used to differ- entiate muscle fibers at least one type of fiber is masked. Also, muscles and/or motor units of different types have been found to develop the same amount of tension while having different speeds of contraction (88,247,256). Close (83) concluded that the observed differences in contractile characteristics are related chiefly to the 32 intrinsic speed of shortening of the muscle fibers and, consequently, that the direct measure of contraction time is of little value when muscle fiber differentiation is sought. Effects of Temperature on Physiological Characteristics Researchers are in general agreement as to the proper muscle temp perature to be maintained while measuring physiological characteristics. Most studies (35,45,57,60,64,65,68,84,88,104,184,186,246,266,293) have been performed with the muscles between 35-3800. Several studies were instrumental in establishing this protocol. Buller st 51, (60), Close and Hoh (86) and Ranatunga and Lumper (284) all found that at tempera- tures below 35-38OC muscles do not contract efficiently. These investi- gators showed that by decreasing the temperature to 10-15°C time to peak tension and one-half relaxation time both were increased. With the decreased temperature maximum tetanus tension was decreased in both "slow" and "fast" muscles. Maximum twitch tension was decreased in "slow" muscle but was increased in "fast" muscle. Ranatunga (285) found that different muscles respond in various ways to a 15°C decrease in temperature. In his study the twitch/tetanus ratio was increased in the flexor digitorium.longus muscle and was decreased in the soleus muscle. The maximum.rate of tension was decreased in the flexor digitorum.longus muscle and was increased in the soleus muscle. Even though these results are generally accepted, a few discrepan- cies have been reported. In two studies maximum tension production was found to be independent of temperature through the ranges 10—36OC (S4) and 25-350C (83). Brown and VonEuler (51) found no relationship between a rise in muscle temperature and an increase in twitch tension. 33 Buchtal and Schmalbruch (55) found the contraction time of "fast" fibers to decrease 10% per degree C and "slow" fibers to decrease 7% per degree C when the muscle temperature was reduced from 32°C to 22°C. Summary of Muscle Characteristics In summary, whether differentiating muscle histochemically, bio- chemically or physiologically, at least three different kinds of muscle fibers have been found. Table 8 presents an abreviated composite analysis of these three fiber types. Development of Muscle Developing muscle also has been examined histochemically, bio- chemically, and physiologically. Thus, this section was subdivided to examine the literature resulting from each of these methods of analysis. Histochemical Evaluation of Developing Muscle The differentiation of muscle into specific fiber types appears at various stages in the development of different species of animals. After 50 days gestation lamb muscle can be differentiated into "slow" or "fast" fiber types via ATPase, and glycogen utilization (PAS) also is distinguishable in these animals (9). Only after 100 days of gesta- tion do the oxidative capacity (SDH) and the glycogenolytic activity (PPL) of lamb muscle differentiate between fiber types (9). The normal gestation period of sheep is 147 days. Beatty gt a1. (30) found "red" and "white" muscle fibers to be differentiated in foetal Rhesus monkeys at 120 days of gestation. At this age muscle fiber size also was different as "red" fibers were smaller than "white". 34 Table 8 Muscle Fiber Characteristics SO FOG FG Oxidative Enzymes High Int-High Low Glycolytic Enzymes Low Int-High High Glycogenolic Enzymes Low Int-High High o-GPD Shuttle Enzymes Low Int-High High Lipid Metabolism High Low' Low Contraction Time Slow Fast Fact Fatigue Index High High Low "Sag" Absent Present Present One-Half Relaxation Slow Fast Fast Time Tension Developed Low Int High 35 Cosmos (90), in studying chicken embryos, found the breast muscle to be low in lactate formation (LDH) and the enzymes of the a—glycero- phosphate shuttle (aGPD) but high in oxidative capacity (SDH). Cepina (80), however, found total LDH activity rises rapidly during the pre- natal development of bovine foetuses. In studying human foetuses, Dubowitz (109) and Strugalska and Fidzianska (314) reported muscle fibers to be differentiated and in a mosaic adult pattern by 26-30 weeks of gestation. The muscle fibers of the newborn rat have been found to be undif- ferentiated (50,178,281,300). Two to three days after birth some differentiation is observable, and by seven days the fibers can be distinguished as "fast" or "slow" with the use of an ATPase stain (49, 50,108,300). Pullen (281) and Dubowitz (108), found that by 14 days of age rat muscle fibers assume a pattern similar to that of the adult animal. Other investigators (49,50,98,178,300) have found that approx- imately one month is needed before the final adult distribution is achieved. Dubowitz (108) and Goldspink (153) found the development of mouse muscle to parallel that of the rat. Goldspink also found that by three weeks of age a relationship is established between the SDH concentration of an individual fiber and the size of the fiber. Smaller fibers con- tain more SDH than do larger fibers. One-day-old pig muscle is differentiable with ATPase and PPL, but clear differentiation into the three fiber types does not occur until the fourth week (89). In the one-day-old pig an extreme range of fiber sizes exists (89). By the third week the muscle fibers are much.more uniform in size, and from the fifth week on the PG fibers increase much more rapidly than do either the SO or FOG fibers (89). 36 Rabbit muscle fibers assume the adult pattern by 14-17 days after birth (235). In kitten muscle, on the other hand, the different fiber types can be distinguished at seven days of age (183). A decrease in the oxidative capacity of some muscles has been found after an initial rise to a peak (90,91,229,234). This reversal of tricarboxylic acid cycle activity appears to be a part of the process of fiber type differentiation. Cosmos (90), in studying chicken breast muscle, found a decrease in SDH activity after the animals were one week old. Cosmos and Butler (91) noted a loss of approximately 60% in the SDH activity of the pectoralis muscle of the chicken at one month of age. By three months of age this muscle consists primarily of PG fibers. In contrast, the soleus muscle of the chicken shows little change in SDH activity after a peak is reached at one week of age (91). Lieberman 35 21. (234) found a 21% decrease in the percentage of red fibers in the diaphragm muscle of guinea pigs after six weeks of age. Kugelberg (229) examined the soleus muscle of rats and found that an increase in SO fibers occurs over the first six months. At one month of age this ratio is 6:1. At five weeks of age the SO fibers comprise 66% of the soleus muscle; at thirty-four weeks of age 93% of the muscle is made up of SO fibers. This apparent gain in SO muscle fibers is not caused by the growth of new SO fibers since Kugelberg (229) and others (236,294) have found the total number of fibers in the soleus remains constant from 1 week through 14 weeks of age. Kugelberg (229) found the rat soleus to have approximately 2,913 fibers and the rat extensor digitorium longus muscle to contain about 3,546 fibers (88). Hooper and McCarthy (201), in studying the mouse, found the biceps brachii muscle to have 1,172 fibers and the tibialis 37 anterior to have 1,694 fibers. Gonyea and Ericson (167) found 8,816 fibers in the flexor carpi radialis muscle of the cat. In comparing the total number of fibers from the left and right muscles of guinea pigs, Karpatti and Engel (217) found differences of 15% between the medial gastrocnemius, 10% between the lateral gastro- cnemius and 25% between the plantaris muscles. Gonyea (168) found no significant differences in the total number of muscle fibers between the right and left flexor carpi radialis muscles of cats. Biochemical Evaluation of Developing Muscle Even though no biochemical studies have been performed on foetal animal muscle, the studies performed on newborn animal muscle show trends similar to those observed with histochemical techniques. Svorvy and Gutmann (316) in studying rats, rabbits and guinea pigs found that myosin ATPase in the extensor digitorum longus muscle increases slightly from birth to adulthood, whereas that in the soleus muscle decreases until the adult level is one-half that of the new born. Hudlicka _e_§ _a_l_. (205) found that oxidative capacity (citrate synthase) and lipid con- tent both decreased after birth, while lactate formation (LDH) had a distinct pattern in kittens by four days of age. In these kitten muscles glycogenolytic activity (PPL) was found to increase continuous- ly after seven days of age, whereas the glycolytic enzyme triosephos- phate dehydrogenase (TPDH) was found to increase only after 21 days of age (205). Enesco and Leblond (130) found DNA to increase with age in the medial gastrocnemius of rats. Hubbard ggngl. (204), also studying rats, found an increase in cytochrome c in the gastrocnemius and soleus muscles of animals between 39 and 87 days of age. The plantaris muscle of this age group showed no change in cytochrome c activity. However, 38 in animals between 87 and 124 days of age, all three muscles showed a decrease in cytochrome c (204). Physiological Evaluation of Develgping Muscle At birth, the contraction and one-half relaxation times of "fast" and "slow" muscles are as nearly alike as they ever become, but even then they are not identical (52,81,242). After birth the contraction time of "fast" muscle has been found to decrease (52,57,82,83,l77,178, 184,242) while that of "slow" muscle has been found to increase (57,177, 178,184,242), stay the same (82), or even decrease slightly (52,83). In cats, the one-half relaxation time decreases in "fast" muscles and increases in "slow" muscles (242) until the adult values are attained at around six to seven weeks of age (184). Close (81,83) found that the twitch/tetanus ratio decreases during the development of rat soleus and extensor digitorum longus muscles. Buller and Lewis (57) found this ratio remains constant in the develop- ing soleus of the cat while it increases in the flexor hallucis longus muscle. Mann and Salufsky (242) also found that this ratio increases in the tibialis anterior muscle of the cat. A few studies have been performed on animal muscle comparing either young muscle to much older muscle or comparing two different groups of adult muscle. Maximum tetanic tension was found to increase in both "slow” and "fast" muscles of the rat and cat up to 14 to 18 weeks of age (81,242). In comparing the extensor digitorum longus muscles of adult hamsters at 180 and 360 days of age, Montgomery (254) found no differences in contraction time, one-half relaxation time, maximum tetanic tension, or twitch tension. 39 Adaptation of Muscle to Physical Training Differences in muscle fiber populations have been observed in human athletes for many years (3,27,76,92,93,95,128,l60,216,280,321). In general, athletes who participate in endurance events have a high per- centage of SO fibers (3,27,92,93,95,128,160,216,321) and a high oxidative capacity in all fibers (76,92,93,l60). Muscles from athletes engaging in sprint events have been found to have a high percentage of PG fibers (128,216,280,321). Varying results have been obtained in regards to the muscle fiber sizes of these athletes. Anderson (3) studied endurance-trained athletes and found the PG and SO fibers to be the same size while the F06 fibers of these athletes were somewhat larger. Brodal £5 51, (44) reported that muscle fiber diameters were similar in trained and un- trained muscles. Edstrom and Ekblom (128) found the mean cross-section- al areas of "red" fibers to be similar in weight-lifters, endurance runners and control subjects, but "white" fibers were significantly larger in weight-lifters than in the other two groups. Prince 35 31, (280) found both F6 and FOG type fibers to be significantly larger in a group of weight-lifters than in a control group. Within the weight- lifters, the FG and FOG fibers were larger than the SO fibers. No fiber size differences were observed in any fiber type between distance runners and control subjects. Thorstensson E£H21° (321) found "fast" fibers to be larger than "slow" fibers in the vastus lateralis muscles of sprinters and jumpers, whereas Costill st 51. (93) found "slow" fibers to be larger than "fast" fibers in the lateral head of the gastrocnemius muscle of distance runners. Since most of the studies involving human muscle have dealt with the athlete after training, the question remains as to how much the 40 muscle can adapt to a training regimen. Consequently, much effort has been spent on the study of animal models in which data can be collected both before and after training. Several approaches have been taken in studying trained animal muscle. Therefore, this section is broken down into the following sub- sections: (a) the effects of aerobic training, (b) the effects of anaerobic training and weight-lifting, (c) the effects of aerobic- anaerobic training, and (d) a comparison of anaerobic and aerobic training. A few studies on humans have been performed in which muscle has been obtained both pre- and post-training. These studies on humans are discussed with the work on animals where appropriate. The Effects of Aerobic Training_ Many different intensities and durations of aerobic exercise have been used in training animals. Training periods ranging from 15 minutes a day (17,22,324) to four hours a day (41,315) have been used, thus a total look at the aerobic continuum.can be obtained. Differences also have been found in the types of activities used as some animals have been swum (41,79,114,115,157,169,172,189,230,262,315) while others have been run (13,79,119,121,142,143,157,l69,192,200,244,329). Most studies have shown that the body weights of exercised animals increase at a slower rate than do those of control animals during train- ing (103,135,157,172,185,189,203,230,262,305), but several investigators have reported body weight to be unaffected by training (112,142,143,l92, 200,244). Carrow £2 31. (97) and Gordon egwgl, (179) in studying rats that had either been run or swum, found the mean body weight of the runners to be significantly less than that of the controls while the mean body weight of the swimmers was not different from that of the 41 controls. This finding is in disagreement with the results obtained by other investigators who swam.rats at the same intensity and for approxi- mately the same duration but found a decreased gain in body weight (114, 157,172,189,230). Several investigators have shown muscle weight to be unaffected by training (121,157,200,262). In these studies lesser bushbabies were run on a treadmill for six months (121), and rats were either swum.(157, 262) or run (157,200) for seven (157,200) or ten weeks (262). Other investigators, however, have demonstrated a significant effect of train— ing on muscle weight (114,192,203). The animals in these studies were either swum (114) or run (192). Maxwell gt El, (244) and Yamaguchi EEUEA: (329) found that training affects only specific muscles. Maxwell 55,31, (244) trained rats on a treadmill at 30 m/min for thirty to forty-five minutes over eight weeks and found the soleus muscles of the trained animals to be heavier than those of the control animals whereas the weights of the plantaris muscles were not different. Yamaguchi 25 El, (329) allowed rats to run voluntarily in running wheels for 30 days and found the weights of the flexor digitorum longus, tibialis posterior and triceps surea to be significantly affected by the train- ing while the extensor digitorum longus, tibialis anterior, rectus femoris and vastus lateralis were not. Through the use of histochemical techniques alterations in muscle fiber profiles have been observed after an aerobic training regimen is performed. These adaptations include primarily an increase in both glycolytic and oxidative activity in all three muscle fiber types, however, other changes have also been seen. Rats endurance trained either by swimming two-hour sessions twice daily for six weeks (41) or by running on a treadmill for 12 weeks (13), have been found to increase 42 both oxidative (13,41) and glycolytic (41) activity in all three muscle fiber types. Eriksson st 21. (136), working with humans trained on a bicycle ergometer for six weeks, found no change in the contraction time of the vastus lateralis muscle as determined histochemically (ATPase). Glycogen (PAS), however, was stored equally well in all fibers while glycolytic (PFK) and oxidative (SDH and NADH) activities were increased at the end of the training period. Gollnick gt El, (162) also trained humans on a bicycle ergometer at 75% of their aerobic power and found increased oxidative capacity in all muscle fiber types while glycolytic activity was increased only in the "fast" twitch fibers. Edgerton 'gtnal, (119) found higher levels of glycogen (PAS) in trained guinea pigs than in sedentary controls. As alterations in the oxidative and glycolytic activities have been noticed so to have changes in the percentages of the different fiber types. Edgerton £5 51. (121) trained adult lesser bushbabies for six months on a treadmill and found no changes in glycolytic, glycogen- olytic or lactate formation enzymes. However, an increase in the per- centage of FOG fibers along with a prOportionate decrease in the F6 fibers of the tibialis anterior muscle was observed (121). No change was seen in the percentage of SO fibers in this muscle, and none of the fiber type percentages changed in the plantaris muscle. Other investi- gators (13,ll4,115,142,l43,244) found that plantaris muscle adapts to both running (l3,142,l43,244) and swimming regimens (114,115). This adaptation was apparent from an increase in the FOG fibers and a decrease in the PG fibers (13,114,115). Syrovy ggugl. (315) trained rats by swimming them two hours every other day for nine weeks and found a decrease of 11.8% in the 50 fiber population with a subsequent increase in the FOG fiber population of the soleus muscle. The Syrovy study is 43 distinguished by the fact that trainingwas inauguarated at 14 days of age. This early starting age has been recognized by others as a distinct factor (13,114,115,244). In one study on adult humans Gollnick g_t_ 31. (162) trained males for five months and observed that the percentages of muscle fiber types were not significantly altered. Muscle fiber size has been shown to adapt to exercise (77,78,114, 121,142,143,162,200,324). Carrow 35 21, (78) found the mean cross- sectional area of the "red" fibers was increased 24% while the mean area of the "white" fibers was increased 10% in the gastrocnemius muscles of rats which were forced to swim but also could run voluntarily. Gollnick ggngl. (162), in a study of endurance-trained humans, determined that the cross-sectional area of "slow" twitch fibers was smaller than that of "fast" twitch fibers before training while the reverse was true after training. The ratio of "slow" twitch to "fast" twitch fiber areas increased from 0.82 before training to 1.11 after training. walker (324) observed that 15 minutes of daily exercise produced hypertrophy, regardless of the intensity, while 5 minutes produced atrophy in mice that were trained for three and one-half weeks. Edgerton 23 £13 (114) reported that fiber hypertrophy was dependent upon the muscle being examined. In both the soleus and plantaris muscles, FOG fibers were larger in trained lesser bushbabies than in control animals. The SO fibers were enlarged in only the plantaris muscle. The mean sizes of the FG fibers in the plantaris and soleus muscles and the size of the SO fibers in the soleus muscle were not different from those of untrain- ed animals. On the other hand, Faulkner and coworkers (142,143) found the "red" and "white" fibers of trained guinea pigs to be smaller than those of sedentary animals. A possible explanation for this 44 observation is the hyperplasia which has been found in the muscles of endurance-exercised animals (79,117). Even though fiber size seems to be affected by training, Holmes and Rasch (200) found no effect on the number of myofibrils per fiber in rat sartorius muscle. The distribution of the myofibrils within the muscle did suggest a training adaptation as a larger number were found at the origin and insertion of the muscle but not in the middportion. In one training study the capillaries of PG and SO muscle fibers were not affected while those of the FOG fibers were significantly increased (238). Carrow g£_gl, (78) studied rats forced to swim and also allowed voluntary running and found a 4% increase in the capillar- ies per "red" muscle fiber and a 10% increase in the capillaries per "white" muscle fiber. Later Carrow at 51, (79) found no change in the capillarybto-fiber ratio of any muscle fiber type in rats which were allowed voluntary exercise, subjected to short, medium.or long duration endurance running programs, or subjected to a long duration endurance swimming program. Biochemically, several different enzymes and substrates of the metabolic pathways have been studied in relation to their adaptation to endurance exercise (22,39,103,121,136,141,144,158,162,172,l74,182,185, 252,262,272,305,325). Muscle glycogen was found to increase with endurance training in both rats (262) and humans (121). Short g£_§1, (305) found the glycogen concentration in trained rats to be higher in "red" fibers than in "white". Glycogen synthetase, an enzyme of glycogenesis, has been found to increase with endurance training. Glycogenolytic activity after endurance training is not as well defined. Baldwin £5 21, (14) found a slight decrease in glycogenolytic activity. Conversely, Gould and Rawlinson (174) observed no change in the PPL 45 activity of rats when pre- and post-trained muscles were studied. Hexokinase has been found to increase in animals after endurance training (14,22,272). Barnard and Peter (22) and Peter £5 51, (272) found this increase to be approximately twofold. Glycolytic activity, as measured by PFR and glucose-6-phosphate (G—6-P), has been found to be unaltered by training in rats (185) and young boys (136). However, Gollnick £5 51, (162) found a significant training-related increase in PFK in the muscles of adult human males. Hearn and Wainio (190) trained rats and obtained an increase in unit and relative total aldolase activity but no change in actual total activity. Baldwin EE.§E9 (14), in looking at the glycolytic activity of specific muscle fiber types, found an increase in the SO fibers, a decrease in the FOG fibers, and no change in the FG fibers after endurance training. Muscle lactate and the enzyme of lactate formation, LDH, have been shown either to decrease (14,158,305) or to remain the same (136,172, 185) with endurance training. These studies were performed on young boys (136) and rats (14,158,172,l85,305); and the gastrocnemius (158, 185), soleus (l4), adductor magnus (305), biceps femoris (172), vastus lateralis (136), and quadriceps (14) muscles were examined. The oxidative capacity of endurance trained muscle, as measured by SDH (103,121,141,162,185,189,258), MDH (189,252) and citrate synthase (148,189), has been shown to increase in vastus lateralis (121,162), rectus femoris (258), gastrocnemius(103,l4l,148,185,189,252,258) and soleus (252,258) muscles. The subjects for the studies which found an increased oxidative capacity were humans (162), lesser bushbabies (121) and rats (103,14l,l48,l85,189,252). However, the SDH activity in the semimembranosus muscles of endurance-trained lesser bushbabies was found to be no different than that in sedentary control animal (121). 46 Cytochrome oxidase (121) and cytochrome c (121,141,204) were found to be increased in the muscles of endurance-trained lesser bushbabies (121) and rats (141,204). Eriksson gt 51. (136) exercised boys for 60 minutes three times per week, for four months, and found a significant increase in ATPase activity of the vastus lateralis muscle. Increases in ATPase also were seen in the predominantly SO and FOG muscles of endurance-trained rats while ATPase activity in muscles of predominantly FG fiber types did not increase (18,315). Wilkerson and Evonuk (325) also trained rats and found an ATPase increase in the gastrocnemius muscles of animals that had been swum to exhaustion every other day for either six or ten weeks but no increase in rats swum.30 minutes every other day for ten weeks. Edgerton at 31, (121), Hearn and Gollnick (191) and Rawlinson and Gould (287) found no significant increase in the ATPase levels of trained animals. The muscles examined were the soleus (121), plantaris (121), tibialis anterior (121), and gastrocnemius (191). Dohm g£_gl, (101) found that total lipid and cholesterol levels were not changed in endurance-trained rats while free fatty acids and triglycerides were both reduced. LipOprotein lipase was found to increase the greatest amount in FOG fibers, but increases also were seen in the SO and FG fibers of endurance-trained animals (40). Myoglobin, the transporter of oxygen in muscle, has been found to increase in endurance-trained hamster (39) and. lesser bushbaby (121) muscles. Mitochondrial concentration (159) and mitochondrial protein (252) have been reported to be significantly increased while total protein content (185,190) does not change with training. Helander (192) and Dohm ggugl, (102) both found sarcoplasmic proteins to be unaltered by training, whereas Gordon at 21, (169) found a significant increase. 47 Dohm g£_§1, (102) also found no change in the myofibrillar proteins, but Gordon gt_§1, (169) detected a decrease. All of these studies were performed on rats. Edgerton g£_gl, (121), studying the physiological characteristics of the plantaris muscle of lesser bushbabies trained at running, found no change in either the twitch or tetanic tension. Furthermore, there was no training adaptation of the muscle in contraction time, one-half relaxation time, tetanus/twitch ratio, fusion frequency or rate of tension development. Effects of Anaerobic Training and Weight-lifting A search of the literature revealed very few studies dealing with only anaerobic training (258,304,310,320). Thorstensson £2 31. (320) trained students with maximal dynamic leg extensor exercises three times a week for eight weeks and found no changes in the percent- ages of the different fiber types utilizing the histochemical ATPase technique. Muller (258) and Staudte 32 31. (310) did observe changes in sprint-trained animals. Body weight (248,310) and actual muscle weight (258) were both found to decrease in sprint-trained rats while relative muscle weight (258,310) was not significantly affected. The percent- ages of fiber types in the soleus muscle were not altered by sprint training. However, the "fast" fibers of the rectus femoris and gastrocnemius muscles decrease in oxidative capacity (SDH) (258). Standte £5 El. (310) found increases in glycogenolytic (PPL), glycolytic (TPDH), and oxidative (citrate synthase, SDH) activities of both the rectus femoris and soleus muscles of sprint-trained rats. Hexokinase increases also were found in both muscles while creatine 48 kinase increased only in the soleus muscle (310). An indicator of fatty acid oxidation (3-hydroxyacyl-COA dehydrogenase) was unchanged with training (310). In studying the physiological characteristics of these animals, Shield 35 al. (304) found the isometric twitch contrac- tion time decreased while the maximum tetanic tension increased in both muscles. Glycogenolytic activity (PPL) was found to increase in rats which were trained with weight-lifting; however, voluntary running produced a greater increase (225). Cytochrome oxidase was found to rise equally in both the weight-lifting and voluntarily run rats (225). Oxidative capacity (SDH) also was found to increase in both rats (225) and hamsters (202) while decreasing in the muscles of mice (309) trained in weight-lifting. Roy g£_§l, (296) found no change in the percentage of "fast" and "slow" muscles of rats that had been weight-lifting; however, the fiber profiles were more variable in the trained animals than in a group of sedentary animals. An unexpected finding of this study was that the trained animals showed a trend toward enhanced aerobic metabolism. Muscle fiber hypertrophy has been observed in animals trained by weight—lifting (152,155,156,l70). Gordon 25 El° (170) reported that only the "white" fibers hypertrOphy, but Goldspink (152), Goldspink it. 31. (156) and Goldspink and Howell (155) found that all types of fibers increase in size. This increase was estimated to be approximate- ly 30% of the average pretraining cross-sectional area (152) and was greater in the biceps brachii and extensor digitorum muscles than in the soleus muscle (155). Another observation in the muscles of weight-lifting animals has been an increase in the total number of muscle fibers (168,196). Gonyea 49 (168) found an increase of 10-24% in the number of fibers in the flexor carpi radialis muscle of cats trained by weight—lifting. Ho 35 _a_l_.. (196), in studying rats, found small split fibers with normal histo— chemical characteristics, adequate blood supplies and neural innerva- tions. Goldspink and Howell (155) and Goldspink Ethel, (156), however, found no fiber splitting present in hamsters that had weight-lifted. The Effects of Aerobic-Anaerobic Training In some studies, animals have been trained both aerobically and anaerobically (10,11,16,23,24,32,97,100,179,l80,l82,l97,l98,l99,206,257, 268). These studies usually have been designed to consist of endurance running with sprints interspersed throughout the training period. Body weight has been observed to be reduced with this type of training (10, 11,97,197). Absolute muscle weight has been found either to decrease (10,11,197) or to remain the same (24,268) while relative muscle weight has been reported to be unchanged (ll). Barnard g£_§l, (23), using a histochemical NADH technique to differentiate the fibers in the medial gastrocnemius muscle, found an increase in the number of FOG fibers and decrease in the FG fibers. The number of SO fibers did not change. Guy and Snow (180) also found an increase in the percentage of FOG fibers, but they noted that both the PG and SO fiber populations were decreased. The subjects for this study were horses, and six different muscles were analyzed. Muller and VOgell (257) found an increase in the number of FOG fibers in the gastrocnemius muscle; however, both the rectus femoris and soleus muscles showed an increase in the number of SO fibers when an endurance- sprint type of training program was used with rats. 50 This type of training program has been found to increase both glycogenic (11) and glycogenolytic (206) activity as well as the activity of the enzyme hexokinase (16,206). Phosphofructokinase (182) and aldolase (180), both enzymes of glycolysis, were found to increase with training while PK (206), another enzyme of glycolysis, and aldolase (206) were unaltered. The activity of the a-glycero-phosphate shuttle (aGPD) was not changed (198); but LDH, the enzyme of lactate formation, was decreased with training (16,179,180,206). All oxidative enzymes studied (citrate synthase, IDH, ketoglutarate dehydrogenase, SDH, and MDH) increased in activity with training. The respiratory capacity, as measured by NADH (197), cytochrome c (197,198,199) and cytochrome oxidase (11,32,100,l97,l98), of animals trained with the endurance-sprint program was found to increase. Increases also were found in amino acid degradation (glutamate dehydrogenase) (199), fatty acid oxidation (11) and myoglobin concentration (268). Baldwin gt 31. (16) studied homogeneous muscles and found hexo- kinase activity to be increased in all three fiber types. The activi- ties of the enzymes oGPD, PPL, PFK, glyceraldehyde-3-phosphate dehydrogenase and PK increased in the SO fibers but decreased in the FOG fibers. Lactate dehydrogenase activity decreased in both the FOG and FG fibers. Most physiological characteristics of guinea pigs trained with an endurance-sprint program were described as being "unremarkable" (24). These characteristics include time-to-peak tension, one-half relaxation time, tetanic-fusion frequency, twitch/tetanus ratio and rate of tension development. In contrast, an unusually high level of isometric tension was maintained by the trained muscles throughout a 60-minute stimulation (24). 51 A Comparison of Anaerobic and Aerobic Training Recently, studies have been conducted which have utilized separate anaerobic and aerobic training programs, thus a direct comparison between the two types of training is possible. In studying human sub- jects that were endurance trained for 12 months or sprint trained for eight weeks, Sjodin gt 31. (306) found no change in the fiber composi- tion of the vastus lateralis muscle. Total LDH activity was unchanged by sprint training while it was decreased with endurance training. Henderson and Reitman (194) also studied sprint- and endurance-trained humans and found no difference in the relative cross-sectional areas occupied by $0 (TYPE I) and FOG and FG (TYPE II) muscle fibers. Oxida- tive activity (SDH) was found to increase 49% in the FOG and FG (TYPE II) fibers of the sprint-trained subjects and 32% in the SO (TYPE I) fibers of the endurance-trained subjects. Phosphofructokinase, an enzyme of glycolysis, was not changed with either training program, Fitts and coworkers (146,147), studying sprint- and endurance- trained minature pigs, found no changes in the fiber type distribution of the biceps femoris and gracilis muscles following three and seven months of training. Muscle fiber size (146) and myoglobin concentration (147) also were similar in the two trained groups and a group of sedentary control animals. Bagby £5 51. (12) found myosin ATPase, determined both histochemi- cally and biochemically, to be unaltered after 11 weeks of either a sprint or an endurance training program in rats. Roy (295) trained rats and found an endurance program increases the aerobic ability of the plantar flexor muscles while a sprint program increases both the anaerobic and aerobic abilities. These results were based on the histochemical evaluation of contractile elements (ATPase), oxidative 52 capacity (SDH), lactate formation (LDH), and glycogen (PAS) and lipid (SUD) localization. In the same study Roy (295) found that eight weeks of training decreased both body and absolute soleus weights. Relative soleus weight was unchanged by training. Training also reduced the absolute plantaris weight, with the sprint-trained plantaris muscles weighing less than the endurance-trained. The relative plantaris weight was greatest in the endurance-trained rats and least in the sprint-trained animals. Taylor (317) also found muscle adaptation when comparing sprint- and endurance-trained rats. In the medial gastrocnemius muscle, oxida- tive activity (SDH) was increased by a long endurance program.after 12 weeks of training while it was increased by voluntary running after only four weeks. Glycogenolytic activity (PPL) increased in a moderate endurance group after eight weeks of training. Glycogen storage (PAS) increased in control, sprint and swim groups after four weeks, in a moderate endurance group after eight weeks, and in a voluntary running group after 12 weeks. Contractile elements (ATPase) increased in the voluntary running group after four weeks and in the endurance trained group after eight weeks. Similar changes were observed in both the plantaris and soleus muscles. Hickson 25 El. (195) found a decrease in glycogenolytic capacity (phosphoglucomutase) and an increase in oxidative capacity (fumurase) in both endurance- and sprint-trained rats. These changes were observed in the plantaris, soleus, and white area of the vastus lateralis muscles after 16 weeks of training. A decrease also was observed in the LDH activity of the white area of the vastus lateralis muscle after 16 weeks of endurance training. In an eightdweek training program, LDH decreased in both the soleus and the white area of the vastus lateralis muscles 53 of the sprint-trained animals. The vastus lateralis muscles of rats sub- jected to an endurance training program for eight weeks showed a decrease in LDH and phosphoglucomutase while phosphoglucoisomerase, an enzyme of glycolysis, was increased in the plantaris muscles. NOne of the enzyme differences between the sprint- and endurance-trained groups were significant after either 8 or 16 weeks of training. Gordon 25 El. (171) found the body weights of rats trained by endurance running and weight-lifting to be significantly lower than those of sedentary control animals. Myofibrillar protein concentration also was decreased with endurance training while sarcoplasmic protein was increased. With sprint training the opposite results were observed. Myofibrillar protein concentration increased and sarc0plasmic protein concentration decreased. CHAPTER III METHODS AND MATERIALS Experimental Animals Twenty-seven random-bred male Syrian hamsters were used as experi— mental anaimals for this investigation. These animals were obtained in four shipments over four months. All animals were provided an environ- mental adjustment period before the treatment was commenced. Initiation of the treatments were begun when the animals were 35 days of age. Treatment Groups Upon arrival all animals were randomly assigned to one of three treatment groups: (a) zero-week control, (b) 12~week sedentary control, and (c) lZ-week high-intensity endurance swimming. The zero-week control group was sacrificed at 35 days of age and thus designated as the pretreatment control group. The lZaweek sedentary control group remained in their cages throughout the duration of the experiment and, therefore, served as a post-treatment control group. The 12~week high- intensity endurance swimming group was exercised five days a week for 12 weeks. A progressive training program was used with the amount of activity gradually increased up to the 37th day of training. By the 37th day of training, the animals were expected to complete a daily swim of one hour with 2-3% body weight attached (see appendix A for the complete training program). Each animal was swum in an individual 54 55 cylinderical tank having a diameter of 28 cm and a height of 75 cm (water depth 70 cm). The training program was performed between 1:00 p.m. and 5:00 p.m. All trained animals were weighed daily before being exercised. An animal was not swum if his body weight had decreased by more than two or three grams from.the previous day. Animal Care Previous work in this laboratory has shown that nocturnal animals which are forced to exercise will perform best between four hours before and four hours after the lights are turned off in the animal living quarters. Thus, the lights in the animal quarters were turned off automatically each day at 1:00 p.m. and were turned on again at 1:00 a.m. The animals were housed in sedentary cages (24 cm long by 18 cm wide by 18 cm tall). Throughout the experiment, all animals had access to water and a commercial hamster chow ES libitume Sacrifice Procedures Six sacrifices were conducted over a sixamonth period. The two initial sacrifices involved only zeroeweek animals while the remaining four sacrifices involved animals in the lZ-week groups. The sacrifices were conducted approximately 70 hours following the last exercise session. Only animals considered to be in good health were sacrificed. A performance criterion of swimming 75% of the sessions with weights attached 75% of the time was set for the trained hamsters. Each animal was weighed and then sacrificed by decapitation. The heart and selected muscles of the left and right legs were removed immediately. 56 Left Leg Muscles The left leg muscles were used for physiological characteristics. Since the instrumentation used in obtaining this data are new, explana- tions of both the physiological stimulating system and the exact sacrifice procedures will be dealt with in this section. Physiological Characteristics Data Collecting System The purpose of the physiological stimulating system were: (a) to maintain hamster muscles at a constant temperature (15°C) in Ringer's solution, (b) to stimulate the muscles electrically according to a pre- determined program, and (c) to record the force curves resulting from the applied stimuli. The stimulation chamber (Figure 1) consisted of a plastic dish (8.9 x 7.0 x 7.6 cm, inside) placed within a larger tank (22.9 x 12.7 x 16.5 cm, inside). The inner dish was filled with Ringer's solution, the outer tank with tap water. The temperature of the inner bath was main- tained at 15 i 1°C. In previous work performed in this laboratory it was observed that this temperature was optimal for the contractile program used. Suspended within the inner bath were two stimulating electrodes, two tissue clamps and a gas dispersion tube for bubbling oxygen into the solution. Each of these was mounted on a plastic support (Figure 2) which slid into two slots on the outside of the larger tank. A second pair of slots held the assembly up and forward for ease in calibration and mounting tissue. The tissue clamp arrangement is shown in Figure 3. The lower clamp was stationary and was intended to pinch a tendon. The upper clamp, designed to support a bone chip, was connected through a nylon insulator 57 Figure 1 Physiological Characteristic Data Collecting Systen Stimulation Chamber 58 . '7‘" 4 ‘ 4— '1 VP-n—Ai “I .I Figure 2 Electrode and Tissue Clamp Assembly of the Physiological Characteristic Data Collecting System 59 [ - J TRANSDUCER INSULATING SPACER NYLON 3/32 OM. HOLE J m l.___,:~ ———*|.\‘LT 69 a 34, DIA. I61" »one 1": W 3 —'— STAINLESS STEEL /32 D". 3]. _L .1— © TENOON CLAMP STAINLESS STEEL a 3/32 OIA. HOLE E: EML. *— ROUND EDGES FACES FOR GRIP TO TISSUE/ Figure 3 Tissue Clamp Assembly of Physiological Characteristic Data Collecting System 60 to a force transducer (Statham UC3 plus 5 pound load cell, UL4). The insulator was required to prevent unwanted current paths through the tissue. The transducer was raised or lowered by a micrometer adjust- ment (Figure 2) to account for differences in individual muscle lengths. The electrodes (Figure 4) were made of 24 ga. sterling silver (1 cm x 4 cm) plates which were glued to plastic supports. Wire leads (18 AWG) were soldered to the back of each silver plate using ordinary techniques and solder. The plates were surrounded with a plastic liquid/powder mix: the same material was used to fill the lead holes. The result was electrodes which were mechanically rugged and had no exposed metal portion except the flat silver face. Because unplated silver presents a very poor electrical interface to Ringer's solution, the electrodes were plated with AgCl to facilitate the flow of current between electrode and solution via an exchange of chloride ions. Each electrode was scrubbed with steel wool and placed in a .1 N solution of HCl with a dummy electrode (Figure 5). A current of 50 mA completed the process in 15-30 sec, as was evidenced by a color change to dull grey on the electrode surface. After 20 muscles had been stimulated, each electrode was cleaned and replated to avoid contamination of the surface. The stimulus was applied to the muscle by a technique called mas- sive stimulation. That is, current was passed between large electrodes through the muscle via the Ringer's solution. There was no direct con- tact between the electrodes and the tissue. This method had several advantages: (a) all parts of all available fibers contracted in unison which avoided propagation delays, (b) tissues remained in the physio- logical Ringer's solution at all times, and (c) no mechanical restric- tions were imposed by electrodes attached directly to the muscles. 61 ELECTRODE (tittfia SUPPORT '/e i 3/4 I 25/3 CLEAR PLASTIC LEAD HOLE _. Q (ALL ”/5, OIA.) —u I.- a n- ALL 50053 “/5‘ o 4 ~34; INOT CRITICAL) .. . i x": -. T 7/3 J PLASTIC _/ O- 348 I‘ FILLER _L 0 ° , Hart” :23 LEFT RIGHT MOUNTING BLV 'L 5 Z SOLDER X Is we was LEAD IBELDEN 8522) MOUNTING NOLEs L ELECTRODE Icm : 48m, 249:: STERLING SILVER 37,, DIA. HOLE 3/32 DIA. HOLE (CENTERED) 7N5 2 NOLEs- 5/32" FROM TOP 8 DDTTOIII (DRILL ALL THE my THROUGH. TAP "548 IN FROM EACH END FOR 6°32 SCREW) Figure 4 Electrode Assembly of Physiological Characteristic Data Collecting System 62 amuwhm moHuUOHHoo puma OAAOAAOAUOAOAU Amoawoaowmhsm uom aanewmmm wcAumHm ooouuooam 0....m44m mo mg... 0.30.... 1:; 04m... KOhmamm a Zorromzzoo m._.<.5mz_ "awn—40m moomhomAm >222. owed...“ mm o... moomhown—m m muswwm \ 535 .._ . ..N\_Z.||.T AAAAAAA ;N\. .48. IT moomkomIfi «$33 no 38 5.50 22.230 oze: S. goo... oez. 9a.. ozAAIIIIT 3 «x. .58. mmtl. AAAAAAA A: PIT. I“ >0 _ _ .1... I? 1"" 63 Very high currents (1-4 amps) were required with.massive stimula- tion because most of the current did not actually cross the muscle membranes. In our system, current pulses of 2 amps and 2 meec width were used: the polarity of each pulse (current direction) was opposite to that of the previous pulse so that net ionic flow was zero (Figure 6). This was necessary to avoid damage to tissues and electrodes. The stimulator was custom made. The output stage was very similar to an audio amplifier. The pulse width and amplitude were controlled by analog circuits. Pulse frequency (for tetanus) was determined externally. The stimulator generated one 2-amp, 2~msec pulse whenever a trigger was received from a separate unit, the stimulus sequencer. The function of the sequencer was to apply a fixed set of stimula- tions and rest periods to each muscle. The sequencer also controlled data rates to a digital recorder and generated start and stop signals so that data were not recorded during rest periods. A mixed program of twitches and tetanus at various frequencies was desired to investigate such parameters as twitch/tetanus ratio, degree of fusion during tetanus and comparison of twitch characteristics before and after partial fusion of tetanus. The present stimulation program used 16 contractions. Contraction numbers 1-5 were twitches initiated with a single 2~msec pulse. Between each twitch there was a 10-sec rest with a ldmin rest following contraction number 5. Contraction numbers 6—11 were 2-sec tetanus contractions at frequencies of 10, 20, 50, 100, 200, and 500 pulses per second (PPS) respectively. Again the rest period between contractions was 10 sec with a l-min rest period following contraction number 11. Contraction number 12 was a 30-sec tetanus at 50 PPS designed to show fatigue effects. After a ldmin rest three more twitch contractions, numbers 13-15, were performed. These 64 swumhm woAuUOAAoo moon UHAAAHOAUOAOAQ Amowwoaowmhcm mo meuomo>m3 AcumaseAAm o OADwAm «Sena ASE itmquon 3 02.22554 m4wc< \ ..m<._on_m.. 3v Aos no. was an uuumuo unmosmaaM4m4 s~5.ooo.e amo.~ smm.ams.ooa.~ sauce mse.m~4 smo.~ 4m¢.4em.~mm Huassmos 4 sec. seo.so mom.Hmm.w~ m4 ~om.~s~.me~.s soasuaaxm 4 sec. mmm.m aso.4flm.a a ~m4.s~s.me mass 4044s I 4444 mason: I usuaumoua Hmafls< 4 use. mNm.m aso.4sm.e a em4.s~s.m4 muosuomumuaH 4431M 4 use. Nm4.aH sem.amm.m s mo~.smo.om mass 40444 u 4044 «Hams: nsu. mm4.H msm.44s NH Hsm.mem.h case Hones . usuaumaua Haase< 4 sec. 4H4.¢H s4a.smm.w 4 sms.s~m.mm 4044 0404:: . usuaummus an4e< 4 sec. 44n.ms ~44.H4w.m NN sse.smm.ema maosuoaumunH smsum 4 soc. ooH.m~ aam.mms.~H w mmfi.aem.ma mass 40:44 4 sec. sa~.em mmm.o~m.e4 N sas.ssa.mm 4444 means: 4 sec. s4o.~mo.fl Hom.mem.oeq N ~m~.s~s.ssm geosumoue Hmasa< 4 sec. smo.oo~ sem.~mn.am NH aae.oaa.eso.s muommmm can: moam> 09Ho> oumdvm we moumsvm coaumqum> m a :40: so sum so muuaom mou< Ronda cause: you muoommm Hauuo>o How ooemfiuo> mo mwmhaue< me wanna 87 Most of the interaction effects were significant in the analysis of fiber area. The only non-significant interaction was that of animal treatment with fiber type (P = 0.145). Physiological Characteristics Data Due to the many physiological characteristics which were obtained from each muscle, this section will deal primarily with those compari- sons which were significantly different. Tables 14-21 contain the mean values and standard deviations of each physiological characteris- tic along with the F-values of the planned comparisons among the groups. Table 14 demonstrates that with contraction number 1, the initial 2-msec pulse twitch, the only differences found were obtained in the peak force corrected for either body weight or muscle weight. The SOL muscle peak force/body weight and peak force/muscle weight were both significantly larger in the zero-week control animals than in the 12-week sedentary control animals (P < .05, P < .05; respec- tively). None of the other variables was significantly different among the groups for either of the muscles studied; and, therefore, no training adaptation was apparent from the data obtained with this contraction. The data obtained from the 2-sec tetanus contractions (Table 15) show similar trends in the first two contractions, even though different stimuli were used. With contractions 6 and 7 (10 and 20 PPS; respectively) no differences were observed in the RF muscle; yet, the SOL muscles of the 12-week sedentary control animals produced a greater force than did those of the zero-week control animals (P < .05 for 88 444 a 44 .4444.4 4 44 444 a 44.44444.44 n 44 4444.44 4 4444.444 4444.44 4 4444.444 4444.44 4 4444.444 444444 444 a 44 .4444.4 4 44 444 u 44 .4444.4 4 44 4444.44 4 4444.444 4444.44 4 4444.44 4444.44 4 4444.44 4444444 444444 unwfioz oHom:z\ouuom xuom 444 u 44 .4444.4 4 44 444 u 44.44444.44 u 44 4444.4 4 4444.4 4444.4 4 4444.4 4444.4 4 4444.4 444444 444 u 44 .4444.4 4 44 444 u 44 .4444.4 4 44 4444.4 4 4444.4 4444.4 4 4444.4 4444.4 4 4444.4 4444444 444444 uswfioz moomxuouom swam 444 u 44 .4444.4 4 44 444 u 44 .4444.4 4 44 4444.4 4 4444.4 4444.4 4 4444.4 4444.4 4 4444.4 444444 444 a 44 .4444.4 4 44 444 a 44 .4444.4 4 44 4444.44 4 444.44 44.44 4 44.44 4444.4 4 4444.4 4444444 444444 va oouom xmom wsuaafiam hufimeoueH mumuoouom soozlouom 4444 4443.44 4443144 H Hoaasz doauomuueoo scum ooaamuno moaumwuouooumzu Handwoaoamzsm «H manna 89 Hm>mH no. 454 um 4044444800 unmowwfiawam 4. 444 a 44 .4444.4 4 44 444 n 44 .4444.4 4 44 4444.44 4 4.444 4444.44 4 4444.444 4444.44 4 4444.444 444444 444 u 44 .4444.4 4 44 A44 4 44 .4444.4 u 44 4444.4 4 4444.44 4444.44 4 44 4444.44 4 4.44 4444444 444444 Aommav mafia coaumxmaum mama 0:0 444 a 44 .4444.4 4 44 444 a 44 .4444.4 4 44 4444.44 4 4444.444 4444.44 4 4.444 4444.44 4 4.444 444444 444 n 44 .4444.4 4 44 444 u 44 .4444.4 4 44 4444.44 4 44.444 4444.44 4 44.44 4444.44 4 4.44 4444444 444444 Aommav QEHH coauomuuaoo waaaafizm hufimcmuaH mumucwvmm xmmzwoumm 4444 4443-44 4443144 44444444444 44 44444 90 2!... no. 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"an...n soca.~n “can... -_.... . acne... nano.n~ . anoo.n~ ...ouu. .a.u.- A v .ouo. gas; 05!; sou-no.5 D3603 5003.23 9:38 533-3.: Cogs-10m .33525 9:33 33:35 C353 goo-fonts and. .ooa.~. aooau~_ g..x .oo:.~. aco:-- g._= .o.:-~_ .oo:-~. o :1... 33:03.30 .IOI. 5 hi :3 39:5... ‘4‘. . unlit-Illh ..~io‘n.l. .n: ot|lI-o;-U.Il.. 0:33.33 vengn I9: 35103 nus-«unavnhufi «nu—.33.??— nu 03.» ‘I‘l'..l¢‘il.. a goal... :3 .01:ch Incv"IrDIFu.l .II.PC"& II'—--‘.cIOIOII-l-.Ilnlluuh o.l|.‘nll'arl'll-‘l3- 'II'II.-'I 91 Ana u we .o~cm.o a mv AHH a mu .mm~m.m u mv “HH~.omq H HHHH.HHOH mmm~.~om H Hmoo.mm~H “mum.onm H HHHa.HhmH maaaom AoH a HH .Hooo.o a my Asa a mu .HoHo.o a mv oaom.m¢~ H ooqm.o- Hemo.~c~ H mmom.mn~ HmHm.wHH H wwwa.wm~ mHHoaom mauoum unwwmz odom:z\muuom xmmm Ana u Ha .mohm.o u my AoH a HH.HHmNN.oH u mv Homo.o H om-.o «moo.o H -m~.o mmoc.o H Nomm.o mauflom AHH n ma .hmoo.o n my ahH u we .HHoH.o u mv «ham.o H musm.o m¢on.o H Hm¢m.o hmHm.o H mhmo.o mHHoamm mauuom unwfimz muom\muuom xmmm AmH u my .quw.H u my AHH u Hc.HmHoh.m a my th~.m H Nqom.H~ Hmoh.e H ~mom.o~ mohm.m H HH¢©.~H mamaom AcH . Ha .HHHH.o n mv Ana a He .mnnm.H n my aqm~.mm H aH-.Hm whom.oo H oooH.mc oomN.MH H Naoo.mm HHHoamm mauuum Amy munch xmom wawaawzm muwmamuaH huwuamwmm xmwzloumw anm kuzwuH Hmmzuma NH umnasz cowuumuudbo Baum vmaamuno muaumaumuumumso HmuwwOHOHmhnm 0H «Hana 92 Ho>mH no. can um comaumnaoo uamuamaawam um An a mu .hooH.o u my Am u mu .monm.o a av «mum.mfl H mash.om NmHa.mm H ommm.mw mmma.mm H hon.~OH msoaom AN a mu .ohm~.H n mv An u we .o~¢a.o u my ¢mHm.H H comm.H~ o H ocqm.ma oh¢H.H H comm.mH mHHoamm usuoum Aummv munch uafiom Ham N\H oH HaHom cam aoHH msHa Am a HH .monm.o u my Aoa a HH.HHomn.m u my omma.o H oqu.aH HnHH.m H ooo~.- Homo.~ H ommm.mH mamaom Aoa n Ha .mcoo.o u my Aha a HH .qum.~ u my NHHo.m H mqoo.wfl ~mmm.m H omqo.¢a mnmo.m H mnmm.oH HHHoamm mauuum Aummv macaw m>fiumwmz xmz ou xmmm scum mafia AmH a HH .mmwm.o n my Aoa u HH.H-mm.H u my HNwH.N H haco.o ¢won.m H ¢owo.h OHMH.H H mqoo.m msmHom AHH a mu .owum.m n mv Asa a Hu.Hooow.- u mv HHOH.N H memo.“ ommm.~ H emom.m mmHm.H H «Hmm.H HHHoamm mauuum Aummv xmum ou mafia wawaawSm huuwcmuaH humucmvmm xmmzonMN anz Homsnwa xmozwma AnmsaHHaoov ea oHnme 93 Ana u Ha .mmHo.o u mv flea a Hu.Hooaa.m~ u my oqu.em H hHHN.H¢ mooo.~c H mH¢H.¢m maHm.HcH H m.o~m maoaom AoH u my .mNom.o u my Ana u Hu.Hm~5H.oH u my mmmH.h H mamm.m ann~.m H ~m¢a.m mm-.oH H omom.HH HHHoamm mauoom uswfim3 mHom:z\mouom xmmm AmH a HH .maao.o a mv flea - HH.HHnH~.oH u my HNHo.o H HoNo.o anoo.o H mmHo.o mHmo.o H ono.o mauflom Aoa u my .H~¢~.o n my aha a Hc.H~om~.HH u my omHo.o H Huao.o mHHo.o H mwoo.o ommo.o H Homo.o HHHoaom mauuum unwawz muom\auuom xmom Ana n Hg .mmHH.o u av “0H u Hu.Hmohm.HH n my N0®H.H H HNNm.H mnom.o H Hamo.~ HMHH.H H “NmH.H mamflom Ana a mu .HmoH.o - av Aha u He .ommm.m n my mem.H H msoH.H ONHH.H H oeo~.o Hemo.H H Humo.a mHHoamm mauoum va munch xmam mafiaaaam madmawuaH humucmvmm xmmzloumm stm 333:3 “#3an ma Hanaaz aowuumuuaoo Baum vMGHMuno mowumaumuomumno Handwoaoamhnm NH manna 94 H0>0H no. any um aomwumgaou unwuwwHQMHm a. Ana u Ha .mmmm.H n my “ea a HH.HHHNH.H a my o~n~.me H Noam.mHH mmq¢.- H nmqo.HoH mm-.we H mHHN.mo~ mamHom flea a mu .omnm.~ a mv Asa n HH.HHmHm.cH u my -H¢.~a~ H HHho.~h~ Homo.oom H memo.an ewec.~m H mNHm.OHH mHHoaum mnuumm Aommav mafia doaumxmamm mama ago And a we .NNHo.o a my AoH u HH.H~m¢m.mH u mv HNNH.o~ H oooo.n~H mo-.HH H onem.m~H mmoo.mH H omNH.HoH mamaom Rea a mu .m¢¢o.¢ u my Aha u Hu.HoHHq.m n my whoo.mH H nmm~.Hm «mmm.H~ H mame.ca mmHH.~H H oooo.mHH HHHoamm msuuom Aoomav mafia aowuomuuzoo waaaafiam magmaMuaH humuamv0m xmmsloumn anm Hmosnua HomszH AvmanfiuaouV NH manna 95 Ana u «H .~¢~¢.o a my AHH u HH.HHmmm.m u mv Humo.omm H Noam.oom H~o~.~nm mHmm.HaoH moc~.omH H omwo.~mma mamaom AHH a mu .mm¢m.¢ u my aka a mu .mH-.~ u mv oqu.cw H HHum.m~ osmH.Hm moo¢.wq m~m~.wm H momm.Hm HHHoaHm msuoum unwwoz oaom:z\muuom xmwm And a mu .HO¢H.o n my AoH u Hv.Hnomo.HH u mv Vnwuo.o H HHoH.o muco.o hmH~.o m~eo.o H «Nam.o msoHom AHH a HH .mem.o a my AHH a H” .mmeo.q u my owha.o H HNmH.o omHH.o HHOH.o «coa.o H noom.o HHHoaum mauuum unwfios hvom\mouom xmmm AmH a mu .mmmm.H a my Ava u HH.H¢HH¢.H u my Hmmo.h H MHHH.mH mmme.e mham.m~ Hoam.m H OOHm.oH mamHom AoH a mu .mnHN.o a mv Aha n we .mmmo.o u my Hmmm.ma H ooaw.mH oomo.~a mmm~.oa mmmm.m H mmcm.m mHHoaam mauoum Amy munch xmwm wawaafiam huamcmuaH Auducmvwm xmm31ouww ame soozw~fl Hmmzme 0H umnasz cowuomuudoo scum vmawwuno muwumfiuouumumnu Handwoaoamhsm mH HHHHH 96 Hw>mH no. mnu um aomfiumnaoo uaduamacwwm « fie - Ha. mmeo.mm H momm.m~a «HHHH.o u my Am a mu auom.mm H mmnq.m- .Hmth.ma a av Hmwm.mH H omaq.caa mamaom Am a HH.HHH¢©.HH n mv o H oomo.~e wo~o.o H onwo.mH HHHoamm mauumm Aummv munch uaflom Ham ~\H ou HaHom can aoHH maHe Ac a my .mom~.o u mv Am a HH .mHo~.o u my mmmm.o H ~¢Hm.qa mmu¢.m H mmo~.~a o~qw.~ H ommm.ma msaflom Am a HH .HH¢¢.H . my “NH a HH .mnmm.a u mv mo~m.m H mmm¢.o~ «Hoc.o H ooHN.H~ HmsH.m H omom.~H HHHoamm mauomm Aommv macaw m>Humwmz an: on 3mmm scum mafia Ana n ma .mmeh.o a mv Aoa u Hu.HmmHm.mH n mv mmHH.~ H mmo~.m momq.q H HHNm.oH wooH.H H ommH.m msmflom AoH n ma .mmHo.o u my Ana u HH.HHH¢m.wm u mv NHHw.~ H MHHH.~H omHH.m H cmam.~a ~o~e.a H omqo.m mHHoamm mauumm Aomwv munch xmom ou mafia wnwaaaam muumcmucH humunmvmm xmwzwouom :mH: xmmzwufi Homsumfl AumsaHHnouv ma manna 97 Ana a mu .Haom.o_umv AoH u an .HHoo.o n my mooa.o H mumm.o HHuo.o H «mom.o mnqo.o H mqom.o mamaom A5 a Ha .Hmmo.o u my AoH a mu .o-¢.o u av Nmmo.o H owom.o emOH.o H NNHm.o mamo.o H sqmm.o mHHoamm asuuum MH Hmnasz aOHuumuucou Hm xmz\Hm owmuo>< AmH u «a .momo.o a mv AoH u mu .Hhoo.o u my meme.o H nmhm.o omno.o H o~cm.o Hmoo.o H mqom.o mauaom AoH n ma .hmqo.o a my AHH a mu .oon.o u my mHmo.o H naum.o «Hmo.o H mwmm.o mmco.o H mwmm.o HHHoamm mauoum H nonaaz aOHuomuuaoo Hm xdz\Hm ammum>< AnH a mu .hmH~.o u mv AHH u HH.HHHom.HH a my mhmo.o H amho.o m-o.o H mmmo.o ommo.o H HmHH.o mamaom AHH a ma .HQHH.o . mv HRH u Ha .5¢O¢.o u mv onmo.o H mm-.o oomo.o H mmo~.o mqmc.o H m~H~.o «HHoaum mauoum Aw\uwma\wv MH w H mQOHuumuuaoo Am x QEHH cOHuomuucoov\AOOH x munch xwmmv waHaaHam huHmcouaH annuamvom xmmzlouMN ame Hoozme Hum: uNH vwmmm mHumsz mo mmuammmz HmUHwOHOHmmnm mH mHnma 98 Hm>wH mo. ofiu um comHummaoo unmoHMHame t AHH u Hg .Hem~.o u my HHH - Ho .Nowm.m u mv mhoa.o H oom~.o- Nemo.o H Hwo~.ou mmmH.o H omNH.on mHHoaom mauoom mam AmH . He .owmo.o . mv AHH . Ha .mHHo.o u my omHo.o H maHo.o mmqo.o H on~o.o ammo.o H Humo.c msmHom AoH a HH .moom.o . mv Aha - He .oemH.o u my mHHo.o H mmHH.o hoHo.o H Hmho.o «Noo.o H oomo.o HHHoamm msuoum n umnaaz GOHuuwuudoo M\m N GOHmsm AnH u Hy .mHmm.o I My AHH n mu .Nmm~.o u mv HmHo.o H ammo.o ammo.o H oumo.o Hooo.o H camo.o maoaom AoH n Ho.Hoomo.m u mv AHH - mu .MHmw.o n mv HH-.o H oqmm.H oomq.o H memm.o Hawm.o H mmm~.o HHHoamm msuoom o umaaaz doauumuudou M\m N GOHmsm wdHaafism muHmcmucH hHMuamwom x003 Iowan :me Homzwma How: -NH AvmaaHu:00v ¢H mHamH 99 Ana n ma .~m~m.o u mv AHH u we .mmhm.m a my ~mqm.~om H Hoo¢.ohfla onam.a~m H mme~.nmma m~m~.Hm~ H MHHN.NHHH msoHom flea u Ha .mqmo.o u mv AHH - HH .Hmmo.H - my «Hem.mmm H muoo.ehq mnmo.o~m H H¢oo.m~m smhm.~wfi H mNHH.owm mHHoamm mauumm uanmB mHum=z\muuom AnH a mu .mmmH.o u my AHH . HH.HHHH©.HH u my mwao.o H onN.o wHoo.o H ¢m0~.o ~m¢o.o H Hoom.o mamHom AoH a mu .mhmH.o - my Aha u Ha .mmhm.o u mv momh.o H «Hma.o ommm.o H H¢~H.H o-q.o H nmmw.o HHHoaum mauuox uanmS mvom\wunom AmH - Ha .mme.H u mv AHH - Hv.HmHmo.~H n my omHm.m H mmeq.m~ ow-.o H ome~.m~ amsa.m H oo¢q.mH mamHom AoH a He .mmhn.o n mv HRH u Hw.HmmmH.m a mv mmHo.H~ H emom.wm Hamm.OH H como.aHH Homm.ofl H ommm.mH HHHoaam mauoum va Amv vouch waHaEH3m muHmcmuaH manucwvmm meBIoumN anm somzuua Hamsuma ON mHan Suwamuum mHuw:z mo mmusmmmz HmonOHOHmhnm 100 HH>HH mo. «nu Hm comHumaaou unonMH=MHm % AmH a mu .oNHo.o u av AQH n Hc.Hmmmm.~H u my mmmo.o H mHHH.o meqo.o H «hofl.o Hmmo.o H HHmH.o maoflom AoH n ma .Humm.e u my AHH . Ha .meo.o a my Hmmo.o H unm~.o m~oo.o H HNHH.o Namo.o H ooo~.o mHHoamm msuoum Am\mv maamuma\suHHaa wddaaHBm huumdMuaH hu~uamvow 3&0310H0N anm xuozme xmm31~H AumaaHuaoov cu manna 101 Ana I He .NHHH.~ a mv ommo.o H quw.o AHH a HH .Nwoo.o a mv mnmo.o +l «hwo.o HHH a mu .oomm.o n my memo.o H wmmm.o AeH I Hu.Hmmm~.NH u mv quo.o H HmnN.o AmH n Ho .Huhm.o a mv maco.o H Numw.o anH.o H Hmmo.o N¢mo.o H mnmm.o mmoH.o +l nmmo.o AHH u HH.H~HHH.H n my Nqu.o Aha u Hc.HwHom.o a my mOHH.o AoH I mv.«vHON.NH I mv moHo.o “NH n Hu.HHooo.HN u mv mNNo.o AHH u mu.Homem.m a mv qum.c mawHom +l mmac.o mHHQamm mauuum NH Hmpadz QOHuuwuucoo I munch xmmm ou munch van mo OHumm +l om¢a.o maoHom +| amom.o mHuoamm msuuum :OHuumHucoo com on I OHumm munch xwwm +l owNH.o H Hmme.o cnmm.o H meN.o omnq.o H mmmN.H msoHom AcH u Ha .Nnm~.H a mv Aha u Hu.Hhocu.mo a mv mmNo.o H weqo.o HNmo.o H oqNo.o ono.o H «NmH.o mHuoawm mauumm :OHuumuuaoo nouHse I OHumm venom xaom waHaaHam huHmaouaH muwuaovmm xmwBIoumN ame xmszNH Humzwmfl mucmumHmmm mawHumh HMHsumsz mo HN anmH mmuammmz HmUHwOHOHmmcm Hm>oH no. «nu um :omHummaou unaonchHm H 102 AHH u «H .oeoq.o u mv AmH u HH.H~H¢H.HH u mv HmoH.o H HonH.o mHoH.o H oHHH.o momH.o H Hneo.o mauHom AmH u Ha .mmqo.q n my AHH u HH.HHHOH.mm . my mmmm.o H owom.o Howm.o H mwnH.H m~e~.o H oo~o.~ HHHoamm mauuum 0H umaaaz GOHuomuuaoo I munch xwwm mo mmoq uawuumm AmH a HH .mmHH.m a mv AHH a mu .ammH.m u my oOOH.o H mm-.o onH.o H «ohm.o mHoH.o H m~m~.o maoHom AHH u «H .HHHo.H u my AHH a HH .mmqm.o u mv ommm.o H «Hom.H mooq.o H omHo.~ HHHH.o H mHH~.H mHHoaam mauoam NH umaapz aOHuumuudoo I munch xmmm mo mmoH uamuuwm AHH a HH .nqom.o a my AmH u Hc.Hmomm.om a my HoHo.o H HHHm.o om~o.o H mHHm.o Homo1o H ommm.o mamHom AmH a HH .Hmwm.m a my AHH u HH.HH~mo.wo u av wmso.o H «Hum.o Heeo.o H mmom.o «Hwo.o H mHom.o mHHoamm mauoum oH umnabz aOHuuwuuaoo I munch xmom ou munch cam mo OHumm maHaafiam muHmamucH humucmvmm xmwzlouoN anm xuosumH Hum: INH AvmsaHuaoov HN mHan 103 contraction number 6; P < .05 for contraction number 7). When analyz- ing the peak force corrected for body weight and muscle weight, the zero-week control animals produced more force (peak force/body weight: P < .05 for contraction number 6; P < .05 for contraction 7; peak force/ muscle weight: P < .05 for contraction number 6; P < .05 for contrac- tion number 7). With the third Z-sec tetanus stimulation, contraction number 9, slightly different results were obtained. Both the SOL and RF muscles from the lZ-week sedentary control animals produced a greater peak force than did the muscles from the zero-week control animals. However, only the SOL peak force corrected for body weight was differ- ent between the zero-week control and the lZ-week sedentary control animals, with the zero-week control muscles producing more force per body weight (P < .05). NOne of the other comparisons with these con- tractions was significantly different. The SOL peak force obtained in contraction number 12, the first 30-sec tetanus, was greater in the lZ-week sedentary control animals than in the zero-week control animals (P < .05) (Table 16). When con- sidering body weight, the SOL peak forces were reversed as the zero- week control animals had a greater peak force/body weight than did the 12-week sedentary control animals (P < .05). The time to peak force was faster for both the SOL and RF muscles in the zerosweek control animals than in the 12-week sedentary control animals (P < .05, P < .05; respectively). The time from peak force to the maximum.negative sIOpe was also faster for the SOL muscles in the zero-week control animals than in the lZHweek sedentary control animals (P < .05). Except for the aforementioned differences for this contraction, none of the variables was found to be different in the RF muscle among the three groups. No 104 differences were observed among the three groups for either muscle in the peak force/muscle weight or in the time from the end point force to one-half the end point force. There was also no difference obtained between the data collected on the 12dweek sedentary control and the 12- week high-intensity endurance swimming animals when analyzing either muscle. As noted in Table 17 with contraction number 13, the second twitch contraction which was recorded, peak force was significantly greater for the SOL muscle in the zero-week control animals than in the lZHweek sedentary control animals (P < .05). There was no difference with this variable when measuring the RF muscle. However, peak force corrected for both body and muscle weight was greater for the SOL and RF muscles in the zero-week control animals than in the lZ-week sedentary control animals (peak force/body weight: P < .05, SOL; P < .05, RF: peak force/ muscle weight: P < .05, SOL; P < .05, RF). The contraction time of this twitch was less for both the lZ-week sedentary control animals' SOL and RF muscles when compared to their zero-week counterparts (P < .05, SOL; P < .05, RF). The one-half relaxation time of the SOL muscle was faster in the 12~week sedentary control animals than in the zero-week control animals (P < .05) but slower for the RF muscle in the lZ-week sedentary control animals than in the zero-week control animals (P < .05). .All of the comparisons between the lZdweek sedentary control animals and lZ-week high-intensity endurance swimming group were not significantly different. For the SOL muscle, peak force for contraction number 16 was great- er in the lZ—week sedentary control animals than in the zer0dweek con- trol animals (P < .05); however, peak force corrected for body and muscle weight was greater in the zer0dweek control animals than in the 12~week sedentary control animals (peak force/body weight, P < .05; 105 peak force/muscle weight, P < .05) (Table 18). None of these variables was different between the two groups when the RF muscle was tested. The time to peak force for both muscles was less in the zeroeweek con- trol animals than in the lZHweek sedentary control animals (P < .05, SOL; P < .05, RF). While the SOL and RF times from peak force to maximum negative slope were not different among any of the groups, the time from the end point force to one-half the end point force was short- er for both muscles in the zeroaweek control animals than in the 12- week sedentary control animals (P < .05, SOL; P < .05, RF). This time was also shorter in the SOL muscle from the lZdweek high-intensity endurance swimming animals than in that from the muscles of the lZ-week sedentary control animals (P < .05). This was the only training adapta- tion apparent from this contraction. Other than the twitch contraction times already mentioned, only two measures of muscle speed were found to yield significant differences among the groups (Table 19). SOL muscle contraction time corrected for tetanus peak force was found to be faster in the 12dweek sedentary con- trol animals than in the zero-week control animals (P < .05). There was no difference with this variable for the RF muscle between these two groups. Another indicator of speed, the development of fusion during the first 2-sec contraction (contraction number 6), showed that the RF muscle developed fusion in the lZ-week sedentary control animals faster than in the lZ-week high-intensity endurance swimming animals (P < .05). None of the other contractile characteristics were significantly differ- ent between the lZ-week sedentary control animals and the 12-week high- intensity endurance swimming animals. The measures dealing with strength, defined as the largest peak force obtained from contraction numbers 6, 7, 9, 12, and 16, were found 106 to be significantly greater for both muscles in the lZ-week sedentary control animals than in the zero-week control animals (P < .05, SOL; P < .05, RF) (Table 20). When this peak force value was corrected for body weight, the SOL muscle was still stronger in the 12-week sedentary con— trol animals than in the zero-week control animals (P < .05); however, no difference was observed with the RF muscle. Peak tetanic force corrected for muscle weight was not different for either the SOL or the RF muscles when comparing these two groups of animals. The last measure of strength the twitch/tetanus ratio, was found to be greater for the SOL muscle in the zero-week control animals than in the 12-week sedentary control animals (P < .05). Again, this measure was not different be- tween the two animal groups when the data obtained from the RF muscle was analyzed. Apparently, there was no significant adaptation to the exercise regimen as demonstrated by these strength measure with either muscle. Table 21 contains the results of the fatiguability measures. Most of these measures were found to be significantly different between the muscles of the zero-week control animals and those of the 12-week sedentary control animals. Both the twitch and 30-sec contractions were found to be less fatigued in the muscles of the zero-week control animals as determined by the ratios of contraction numbers 13:1 and 16:12 (twitch: P < .05, SOL; P < .05, RF: 30-sec: P < .05, SOL; P < .05, RF). The only significant fatigue adaptation to occur with training was in.the peak force ratio for the 30-sec contraction (16:12) of the RF muscles of the trained animals which was larger than that of the lZ-week sedentary control animals (P < .05). In the 30—sec tetanic contractions, the ratio of end force to peak force showed greater fatigue to be characteristic of the zero-week control animals. This observation was 107 true for both the SOL and RF muscles and for both 30-sec contractions (contraction number 12: P < .05, SOL; P < .05, RF: contraction number 16: P < .05, SOL; P < .05, RF). The percentage loss of peak force was not different among the muscles of any of the animal groups for contrac- tion number 12, while the percentage loss in contraction number 16 was greater for both muscles in the zerosweek control animals than in the 12-week sedentary control animals (P < .05, SOL; P < .05, RF). Discussion The data concerning the body and muscle weights is in concert with that reported in the literature. That is, the weights increased with age, but these increases were retarded when a physical training program was imposed (108,114,141,157,185,189,203,230,262,305). Since no significant differences were found in the relative muscle weights, it appears that the SOL and RF muscles and the body developed at the same rate. The percentages of "slow" and "fast" twitch fibers did not change in the SOL muscle with either growth or physical training. In the deep area of the RF muscles, however, changes in fiber type percentages were observed. In this area 13.3% of the fibers were "slow" twitch in the zero-week control animals, 20.6% of the fibers were "slow" twitch in the 12-week sedentary control animals, and only 9.0% of the fibers were "slow" twitch in the lZ-week trained animals. Since the superficial RF muscle contains only fast fibers no changes in fiber percentages were observed in this muscle area. The data obtained from the SOL muscle is in agreement with that in the literature which found no change in the percentage of "slow" and "fast" twitch fibers with growth (49,50,98,108,173,300). Thus, the data 108 obtained from the deep area of the RF muscle disagrees with this infor- mation. However, the data from the deep area of the RF muscle is in agreement with the literature which found an increase in the percentage of FOG (FGL-FGgL) muscle fibers after a period of physical training (12, 114,115,121,l38,315). In these studies the increase in the percentage of FOG fibers was paralled by a decrease in the FG fiber population (13, 114,115,121,138) or SO fiber population (315). In the present experi- ment the increase in the FGL-FGgL fiber population was proportionate to the decrease in the F, SGL-SGgL and SL fiber papulations. In accordance with the literature, the FGL-FGgL (FOG), FG-FGg (FG), and SgL (80) fiber types encompassed most of the muscle fibers profiled. However, as was previously noted other fiber types were observed which deviated from the three main fiber types. In the zero-week control animals 6.7% of the SOL fibers were dif- ferent from the three main fiber types (5.6% "slow", 1.1% "fast"). This percentage was 8.3 for the muscles from the lZ—week sedentary control animals (3.3% "slow", 5.0% "fast") and 18.9 for those from the trained animals (12.2% "slow", 6.7% "fast"). It would appear that age slightly enhanced the ability of both the "fast" and "slow" fibers of the central area of the SOL muscle to metabolize glycogen anaerobically. Endurance training, on the other hand, caused an increase in both anaerobic and aerobic glycolytic metabolism of the slow fibers and a slight decrease in these activities of the fast fibers. In the superficial area of the RF muscle, the fibers not classified as FGL-FGgL (FOG) or FgL (FG) accounted for 2.4% of the fibers in the zero-week control animals, 0.6% of the fibers in the lZHweek sedentary control animals, and 11.7% of the fibers in the trained animals. With increasing age it would appear that this muscle area becomes more 109 oxidative while retaining its glycolytic activity. However, with train- ing the amount of anaerobic and aerobic glycolytic activity was reduced while the oxidative capacity was not necessarily increased. The ability to store glycogen was decreased in these fibers. The deep area of the RF muscle reflected the greatest alteration in the percentage of fibers varying from the three main fiber types. In the zero-week control animals, 4.8% of the fibers were found to deviate from the three main fiber types (2.4% "slow", 2.4% "fast). This per- centage increased to 32.5% of the muscle fibers in the lZ-week sedentary control animals (12.5% "slow", 20.0% "fast") but decreased to 7.2% of the observed fibers in the lZ-week high-intensity endurance swimming animals (0.6% "slow", 6.6% "fast"). The age-related trend in this muscle area was for the "slow" fibers to have an increased ability to metabolize glycogen both anaerobically and aerobically, while the "fast" fibers had a reduced ability to metabolize glycogen aerobically. A reduction in the ability to store glycogen also was observed in both the "fast" and "slow" muscle fibers. With training the anaerobic and aerobic glycolytic ability of the "fast" mugcle fibers was increased, but not enough to achieve the level observed in the zero-week control animals. The only training effect on the "slow" fibers was a decrease in the capacity to metabolize glycogen. Glycogen storage was increased in both fiber types with training. The apparent shift in fiber profiles induced by age disagrees with the literature reviewed in which muscle fibers were found to have adult characteristics by at least three to four weeks of age (49,50,89,90,108 153,178,183,235,281,300). None of these earner studies, however, was performed on hamsters. It should be noted that the zero-week control 110 animals in this study were five weeks old, well past the age when most animal muscle is developed. There appear to be two separate possibilities to explain why the "fast" muscle fibers of the trained animals generally declined in their ability to metabolize and store glycogen while the "slow" fibers gener- ally increased their capabilities of anaerobic and aerobic glycolytic metabolism. Initially, the tendancy could be an adaptation to the aerobic endurance training program to which the animals were subjected. This type of training program might stimulate the "slow" fibers to a greater extent than the "fast" fibers; thus the corresponding increase and decrease in glycolysis. However, similar training programs have been shown to increase the glycolytic activity in all three muscle fiber types (41,136,162,3OS). Thus this explanation does not seem probable. Second, the decline in the glycogen storage ability of the "fast" twitch fibers of these muscles could be attributed to the mode of sacrifice. In this study, immediately after decapitation the animals could be observed to go through a few spontaneous contractions which could have affected the stored glycogen levels. Marquez-Julio and French (243), in their study comparing muscles from rats which were either decapitated or anesthetized with pentobarbitol, found that the glycogen content was 40% less in the muscles of the decapitated animals. This would seem to be a possible explanation for the decrease in glycogen storage capacity in these animals, as similar exercise programs have been shown to in- crease the amount of glycogen stored in the trained muscles (119,136, 262). However, since all animal groups in this study were sacrificed by the same techniques this occurrance does not appear to be the primary cause for the lower glycogen levels in the fast twitch fibers of the trained animals. . lll Whether or not the oxidative capacity of the muscle fibers increased is difficult to discern from the present data. The deep area of the older animals RF muscle decreased in the percentage of FGL-FGgL muscle fibers and increased in the percentage of FG-FGg muscle fibers. This data is in agreement with that of earlier investigators who found the oxidative capacity of muscle fibers to decline with age (90,91,229,234). However, the animals in this study were older than the animals used in most of the previous studies. In the superficial area of the RF muscle the apposite trend was seen as the percentage of FGL-FGgL muscle fibers increased with age and the percentage of FG-FGg muscle fibers decreased with age. Since this observation is in conflict with the literature it is difficult to explain the exact nature of this reversal. With endur- ance training the percentage of FGL—FGgL fibers increased in the deep area of the RF muscle and decreased in the superficial area of this muscle. Since with similar training programs an increase in the oxida- tive capacity of the muscle has been found (13,41,103,115,121,136,l4l, l49,l62,185,189,252,258), the explanation for the decrease in FGL-FGgL muscle fibers in the superficial area of this muscle is not readily apparent. Whether or not the other fiber types have increased or de- creased in their oxidative capacity is difficult to analyze from the present data as the fiber profiles generally do not account for changes in the NADH staining intensity. As can be seen by examining the table of the metabolic characteristics for each type of fiber (Appendix B), fibers could have low, moderate, or high levels of oxidative activity and still have the same metabolic profile. Thus, an increase or a de- crease in the oxidative capacity of a muscle fiber might go unnoticed with the present scheme of fiber profiles if all of the other metabolic characteristics of the muscle fiber remained the same. 112 The data obtained on the muscle fiber area appears to be in agree- ment with the literature concerning the effect of growth and develop- ment, but in disagreement concerning the effects of training. In previous studies (78,121,162,324) trained animals were found to have larger muscle fibers than sedentary animals. In the present experiment this observation was reversed, but is in agreement with the data pre- sented by Faulkner and coworkers (142,143). The phenomenon could be due to the fact that the trained animals' body and muscle weights were less than those of the sedentary animals. The reason why the muscle fiber type had no effect on muscle fiber size is hard to explain as other investigations have shown the different muscle fiber types to have different muscle fiber sizes (69,72,116,212,214,227,229,24l,288,299,323). The main cause for differences in the physiological characteristics of the muscles appears to be the growth and develoPment of these animals and not necessarily their performance in the high-intensity endurance swimming program. Generally, the lZ-week sedentary control animals' muscles were stronger than those of the zero-week control animals. How- ever, when the effects of either body weight or muscle weight were eliminated, the zeroaweek animals' muscles appeared to be stronger. This is in agreement with the results of Close (81) and Mann and Salafsky (242) both of whom found that peak tension rose with development in rats and cats up to 14-18 weeks of age. In looking at contraction times, the twitch contraction times of the lZ-week sedentary control animals' muscles were faster for both the SOL and RF'muscles. This is in agreement with those investigators who found a decrease with age in contraction time of both "fast" twitch muscle fibers (52,S7,82,83,177,l78,l84,242) and "slow" twitch muscle fibers (52,83). When examining the time to reach.maximum.tetanic tension in the 113 30-sec contraction, the zero-week control animals' muscles were faster than those of the lZHweek sedentary control animals. These results were true with both 30—sec contractions (contraction numbers 12 and 16) obtained on either the SOL or the RF muscle. The larger twitch/tetanus ratio found with the SOL muscle of the zero-week control animals, when compared to that of the lZdweek sedentary control animals, is in agreement with the data presented by Close (81, 83). However, Close also found this relationship to hold true in the rat extensor digitorum longus muscle, a "fast" twitch muscle. No significant difference was observed in the twitch/tetanus ratio of the RF muscle in this study. The muscles of the zero-week control animals tended to be more fatigue resistant than were those of the lZ-week sedentary control animals. This was apparent from both the twitch and 30-sec contractions. In reviewing the literature no information was found dealing with the fatiguability of a muscle before and after a specified aging period. Since the lZ-week sedentary control and lZHweek high—intensity endurance swimming animals were significantly different in only three of the 94 physiological characteristics obtained from the SOL and RF muscles, there appears to be no overall effect of the aerobic endurance training program on contractile characteristics. This lack of a train- ing effect could be explained, at least in part, by the mode of exercise. When animals are forced to swim, many uncontrolable variables can affect the level at which the animals are trained. A few of these variables include: (a) the amount of air in the fur of the animals, (b) the body density of the animal, and (c) the efficiency or lack of efficiency in swimming skills of the animals. Thus the exact degree of physical train- ing achieved may vary greatly among the animals. Of course, it is 114 possible too that these physiological characteristics simply are not altered by an endurance training program, Edgerton gt_§l, (121) trained rats on a treadmill and found no significant differences between the muscles of the trained and untrained animals. The characteristics measured in that study included twitch and tetanic tension, contraction time, one-half relaxation time, twitch/tetanus ratio, fusion frequency and the rate of tension development. CHAPTER V SUMMARY, CONCLUSIONS AND RECOMMENDATIONS Summary The purpose of this study was to determine the effects of growth and development (aging) and physical training on selected histochemical and physiological characteristics of normal hamster muscle. Twenty- seven Syrian hamsters served as subjects in this study. These animals were assigned randomly to one of the following groups: (a) zero-week control (n = 8), (b) lZ-week sedentary control (n = 11), and (c) 12- week high-intensity endurance swimming (n 8 8). Histochemical analyzes of the right leg muscles were performed on the central area of the SOL muscle along with the deep and superficial areas of the RF muscle. Muscle fiber profiles and respective fiber sizes were determined using 30 adjacent muscle fibers from each of the three muscle areas. Contractile elements of the muscle fibers were determined through the use of the ATPase stain. Oxidative capacity of these fibers was demonstrated with the NADH stain, while aerobic gly- colytic activity was shown by the aGPD stain. Lipid and glycogen localizations were determined through the use of the SUD and PAS stains. Physiological analyzes were conducted using the left leg SOL and RF muscles. In these analyzes many physiological characteristics were determined including muscle strength, speed, rate of relaxation, and 115 116 fatiguability. Some values, such as those from the strength measures, were corrected for body and muscle weight. Histochemically, the percentage of "fast" and "slow" fibers did not change in the central area of the SOL muscle or in the superficial area of the RF muscle. In the deep area of the RF muscle, however, a de- crease in the percentage of "fast" fibers was observed with aging while an increase in this percentage was seen with physical training. Metabolically, the ability of the muscles to utilize glycogen anaerobically and aerobically was increased with age. With physical training the "slow" fibers of the central area of the SOL muscle and the "fast" fibers of the deep area of the RF muscle increased in their capacity to metabolize glycogen anaerobically and aerobically. The other fibers, including those of the superficial area of the RF muscle, decreased in their capacity to metabolize glycogen. The ability to store glycogen was decreased with growth and develOpment in the "fast" and "slow" fibers of the deep area of the RF muscles. In the super- ficial area of the RF muscle of the trained animals, the "fast" fibers had a reduced amount of glycogen stored; whereas, both fiber types of this deep muscle area in the trained animals had an increased amount of stored glycogen. Physiologically, most characteristics were found to be affected by aging but not altered by the animals performance in a higheintensity endurance swimming program. Generally, the muscles of the zeroeweek control animals were faster contracting and relaxing and also less fatiguing than the corresponding muscles of the lZ-week sedentary con- trol animals. The muscles of the older animals did develope more absolute force. However, when this force was corrected for either 117 muscle or body weight, the muscles of the zero-week control animals were stronger. Conclusions From the data obtained in this study the following general conclu- sions may be drawn: 1. The metabolic characteristics of the muscle fibers of the central area of the SOL muscle and the deep and superficial areas of the RF muscle were altered by growth and development and by the performance of a high-intensity endurance swimming program. Muscle fiber size increased with age, but this increase was retarded when the animal was subjected to a high-intensity endurance swimming program. The physiological characteristics of the muscles changed greatly with growth and development as these muscles became slower and stronger with age. Animals who were subjected to a high—intensity endurance swimming program did not alter the physiological characteris- tics of their SOL and RF muscles. Recommendations In future studies dealing with the physical training of animals, a training method other than swimming should be used. Such methods could include running, jumping, or lifting regimens - all of which could be quantified. In studies measuring muscle glycogen, the method of sacrifice should not be decapitation. 3. 118 A revision of the muscle fiber profiles used in this study should be made before future studies are carried out in order to more fully incorporate the oxidative capacity of the muscle fiber into the muscle fiber profile. Biochemical studies on comparable muscles should be performed to complete the evaluation of these muscles. Studies involving the histochemical, biochemical, and physio- logical characteristics of single motor units of muscle from trained animals should be performed. "L LIST OF REFERENCES 10. 11. LIST OF REFERENCES Aherne, W., D. R. Ayyar, P. A. Clarke and J. N. Walton, "Muscle Fibre Size in Normal Infants, Children and Adolescents: An Autopsy Study." J} Neurol. Sci. 14: 171, 1971. Al-Amood, W., A. J. Buller and R. Pope. "Long Term Stimulation of Cat Fast Twitch Skeletal Muscle." Nature 244: 225, 1973. Anderson, P. "Capillary Density in Skeletal Muscle of Man." 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C - 0‘ O m APPENDIX B MUSCLE FIBER PROFILE CHARACTERISTICS 145 Table B-1 Muscle Fiber Profile Characteristics - Fast Fibers Low SDH Mod SDH High SDH (L) (n) Y in) ATPase High (D) Low Mud High Low Mud High Low Mud High Twitch Fast aGPD aGPD oGPD oGPD oGPD oGPD oGPD aGPD aGPD (Q (N) (D) (L) 00 (D) AL) (M) (Di High FgL FGL FGL FgL FGL FGL FgL FGL FGL SUD FGgL FGgL FGgL FGgL FGgL FGgL (1.31 High Mud FgL FGL FGL FgL FGL FGL FgL FGL FGL PAS SUD FGgL FGgL FGgL FGgL FGgL FGgL (D) (M) Low F3 F0 FG F PC PC F FG FG SUD FGg FGg FGg FGg FGg FGg (L) High FgL FGL FGL FgL FGL FGL FgL FGL FGL SUD FGgL FGgL FGgL FGgL FGgL FGgL (D) ‘ Mod Mbd FgL FGL FGL FgL FGL FGL FgL FGL FGL PAS SUD FGgL FGgL FGgL FGgL FGgL FGgL (M) 00 Low Fg PC PC F FG FG F FG FG SUD FGg F03 F03 FGg FGg FGg (L) High FL F F FL F F FL F F SUD (D) Low Mud FL F F FL F F FL F F PAS SUD (L) (M) Low F F F F F F F F F SUD Q 146 Table B-2 Muscle Fiber Profile Characteristics - Slow Fibers W Low SDH Mod SDH High SDH (L) (M) (1)) ATPase Low (L) Low Mod High Low 9‘ Mod High Low Mod High Twitch Slow oGPD oGPD aGPD aGPD 'aGPD aGPD aGPD cGPD aGPD (L) (M) iDL (L) (M) (D) (L) (M) (D) High SgL SGL SGL SgL SGL SGL SgL SGL SGL SUD SGgL SGgL SGgL SGgL SGgL SGgL (D) High Mod SgL SGL SGL SgL SGL SGL SgL SGL SGL PAS sun 3ch SGgL SGgL SGgL SGgL SGgL (D) (M) Low Sg SC 86 S SC 86 S SC SC. SUD SGg SGg SGg SGg SGg SGg (L) High SgL SGL SGL SgL SGL SGL SgL SGL SGL SUD SGgL SGgL $ch SGgL SGgL SGgL (D) Mod Mod SgL SGL SGL SgL SGL SGL SgL SGL SGL PAS SUD $ch SGgL $ch SGgL SGgL 5ch (M) (M) - Low Sg SG SG S SC SC S SC SC SUD SGg SGg SGg SGg SGg SGg (L) High SL S S SL S S SL S S SUD (D) Low Mod SL S S SL S S SL S S PAS SUD (L) (M) g r Low 5 3 s s ,2 s f s s s s s SUD ‘ 5 (L) i :