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Morphological quantitative and ultracytochemical studies on the internal membrane systems of normal and… Nahirney, Patrick Charles 2000

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M O R P H O L O G I C A L , Q U A N T I T A T I V E A N D U L T R A C Y T O C H E M I C A L STUDIES O N T H E I N T E R N A L M E M B R A N E SYSTEMS OF N O R M A L A N D M D X -D Y S T R O P H I C M U R I N E S K E L E T A L M U S C L E FIBERS By P A T R I C K C H A R L E S N A H I R N E Y B.Sc, Washington State University, 1990 M . S c , The University of British Columbia, 1993 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF D O C T O R OF P H I L O S O P H Y i n T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Anatomy) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF BRITISH C O L U M B I A January 2000 © Patrick Charles Nahirney, 2000 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Coh^nibia Vancouver, Canada Department DE-6 (2/88) A B S T R A C T Mitochondr ia and the sarcotubular system i n skeletal muscle fibers are specialized intracellular membranes that participate i n the generation of energy (ATP) and i n the regulation of contraction and relaxation. This thesis comprises four parts dealing wi th morphological, histochemical, stereological and biochemical approaches to study these organelles i n muscle fibers of adult normal and dystrophic (mdx) mice. The purpose of this research was to test the hypothesis that the internal membrane systems of skeletal muscle fibers are altered i n the mdx mouse. Mitochondrial oxidative capacities were assessed in muscle fibers of the extensor d ig i torum longus (EDL) , soleus and diaphragm of normal and mdx mice by histochemical and microphotometric methods i n Chapter 1. Relative to controls, significant shifts i n oxidative staining patterns of mdx muscle fibers were observed including a 20% overall lower oxidative capacity i n the mdx E D L and soleus and a contrasting 7% higher oxidative capacity i n the mdx diaphragm. Transmission electron microscopy (TEM) was combined wi th stereology i n Chapter 2 to determine the ultrastructural arrangement and volume density of the sarcotubular system i n muscle fibers i n these muscles. Three fiber types (white, intermediate and red) were distinguished i n the normal and mdx E D L based on mitochondrial volume i n fiber cores and Z line thickness. A mixture of two fiber types, intermediate and red fibers, occupied the soleus and diaphragm. The majority of mdx muscle fibers displayed a similar ultrastructure and disposition of these organelles i n comparison to their normal counterparts w i t h nonsignificant differences i n sarcotubular volume density between normal and mdx fiber types. Degenerative fibers i n mdx muscles exhibited dilated SR, sarcolemmal and myofibri l disruption, and mononuclear cell invasion. High-resolution scanning electron microscopy (HRSEM) was uti l ized i n Chapter 3 to reveal the spatial distribution of mitochondria and the sarcotubular system by extracting non-membranous components of the sarcoplasm i n freeze-i i fractured specimens. Final ly , i n Chapter 4, a cerium-based, ultracytochemical staining method was used for the detection and measurement of the calcium and magnesium-dependent paranitrophenylphosphatase ( C a 2 + - M g 2 + pNPPase) activity associated wi th the sarcoplasmic reticulum (SR) membrane. By T E M , the majority of C a 2 + - M g 2 + pNPPase activity was localized to the terminal cisternae regions of the SR membrane w i t h moderate phosphatase activity i n m i d - A band and I band regions of the SR. Comparative spectrophotometry of reaction media revealed a significant 25% higher phosphatase activity i n mdx muscle tissue when compared to normal muscle suggesting that Ca 2 +-sequestering by the SR is enhanced i n muscle fibers of the mdx-dystrophic mouse. Overall , these data suggest that mitochondria and SR are altered i n the skeletal muscle fibers of mdx mice and point to specific differences i n oxidative capacity and Ca 2 +-sequestering properties of these organelles. i i i T A B L E OF C O N T E N T S Page Abstract i i Table of Contents i v List of Tables v i i i List of Figures ix List of Abbreviations x i i i Acknowledgements xv General Introduction Overv i ew 1 The Skeletal Muscle Fiber 1 Internal Membrane Systems of Skeletal Muscle Fibers 4 Sequence of Events in Excitation-Contraction Coupl ing and Relaxation 7 Skeletal Muscle Fiber Types 10 Duchenne Muscular Dystrophy, the M d x Mouse and Other A n i m a l Models 13 Statement of the Objectives and Hypotheses 17 References 19 Chapter 1 Comparison of Oxidative Capacity i n Skeletal Muscle Fibers of N o r m a l and M d x A d u l t Mice : A Microphotometr ic Study on NADH-TR-S ta ined Cryosections. Introduction 27 Materials and Methods Animals and Tissue Preparation 30 N A D H - T R Staining 31 i v Myofibri l lar ATPase Staining 31 Quantitation and Image Analysis of NADH-TR-Sta ined Sections 33 Results General Anatomy of the E D L , Soleus and Diaphragm 36 Microscopic Appearance 37 Morphometric and Microphotometric Analysis 40 Correlation of N A D H - T R Staining wi th Type I, Ha and l ib Fibers 53 Discussion 71 References 77 Chapter 2 Morphology and Quantitative Ultrastructure of Mitochondr ia and the Sarcotubular System i n Muscle Fibers of the Extensor Digitorum Longus, Soleus and Diaphragm i n Normal and M d x Adu l t Mice. Introduction 82 Materials and Methods T E M Processing 85 Morphometr ic Measurements 86 Results 90 Norma l Morphology EDL 90 Soleus 102 Diaphragm 107 M d x Morphology EDL 117 Soleus 126 Diaphragm 126 Morphometric Analysis of Normal and M d x Muscle Fibers 133 v Discussion 149 N o r m a l Ultrastructure 149 M d x Ultrastructure 154 References 157 Chapter 3 Three-Dimens iona l Ul t ras t ructure of M i t o c h o n d r i a and the Sarcotubular System i n Skeletal Muscle Fibers of Norma l and M d x A d u l t Mice as Revealed by High-Resolut ion Scanning Electron Microscopy. Introduction 161 Materials and Methods 165 Results 167 Three-Dimensional Ultrastructure of Norma l Muscle EDL 168 Soleus 179 Diaphragm 182 Three-Dimensional Ultrastructure of M d x Muscle EDL 189 Soleus 189 Diaphragm 194 Stereo Pairs 203 Discussion 214 References 224 Chapter 4 Use of a Cerium-based Ultracytochemical Method for the Study of Calc ium- and Magnesium-Dependent Phosphatase Act iv i ty i n the Sarcoplasmic Reticulum. v i Introduction Calcium-Transporting Enzymes 229 Calcium Balance and Muscular Dystrophy 232 Cerium-based Ultracytochemistry 233 Materials and Methods A n i m a l s 238 Cytochemistry 238 T E M Processing 240 Results 241 Discussion 259 References 265 General Discussion 272 References 280 Appendix 282 v i i LIST OF T A B L E S 1. Mean cross-sectional areas and N A D H - T R optical density values for total muscle fibers examined i n the E D L , soleus and diaphragm of normal (N) and dystrophic (Mdx) mice. 41 2. Fiber type proportions, mean cross-sectional areas and N A D H - T R optical density values for type Ila and type l ib fibers i n the E D L of normal (N) and dystrophic (Mdx) mice. 54 3. Fiber type proportions, mean cross-sectional areas (um 2) and N A D H - T R optical density values for type I, type Ila and type l ib fibers i n the soleus of normal (N) and dystrophic (Mdx) mice. 58 4. Fiber type proportions, mean cross-sectional areas (urn2) and N A D H - T R optical density values for type I, type Ila and type l i b fibers i n the diaphragm of normal (N) and dystrophic (Mdx) mice. 61 5. Morphometr ic measurements of mitochondria , SR, T-tubules, l i p id and Z line thickness i n white, intermediate and red fibers of the normal and mdx E D L , soleus and diaphragm muscles. 143 v i i i LIST OF FIGURES 1. L o w magnification views of NADH-TR-s ta ined cryosections of the E D L , soleus and diaphragm from normal and mdx adult mice. 39 2. Frequency distribution of muscle fiber cross-sectional area for total fibers examined i n the normal and mdx E D L , soleus and diaphragm. 43 3. Box plots of the optical density of NADH-TR-s ta ined muscle fibers i n the normal and mdx E D L , soleus and diaphragm. 46 4. Transverse serial cryosections of the normal and mdx E D L stained conventionally w i th H & E and histochemically to detect N A D H -TR and mATPase after acid preincubation. 48 5. Transverse serial cryosections of the normal and mdx soleus stained conventionally w i t h H & E and histochemically to detect N A D H - T R and mATPase after acid preincubation. 50 6. Transverse serial cryosections from the costal region of the normal and mdx diaphragm stained conventionally w i th H & E and histochemically to detect N A D H - T R and mATPase after acid preincubation. 52 7. Frequency distributions of fiber cross-sectional area for the normal and mdx E D L , separated into type Ha and l ib fiber types. 56 8. Frequency distributions of fiber cross-sectional area for the normal and mdx soleus, separated into type I, type Ha and type l i b fiber types. 60 9. Frequency distributions of fiber cross-sectional area for the normal and mdx diaphragm, separated into type I, type Ha and type l ib fiber types. 63 10. Box plots of the optical density of NADH-TR-s ta ined fibers i n the normal and mdx E D L , soleus and diaphragm separated into type I, type Ha and type l ib fiber types. Whole fiber measurements. 65 11. Box plots of the optical density of NADH-TR-s ta ined fibers i n the normal and mdx E D L , soleus and diaphragm separated into type I, type Ha and type l ib fiber types. Fiber core measurements. 67 12. Example of a myodegenerative region i n the mdx soleus stained conventionally wi th H & E and histochemically to detect N A D H -TR and mATPase after acid preincubation. 70 ix 13. Low power transmission electron micrograph (TEM) of a transverse section of the normal E D L . 93 14. High magnification transverse view of a white fiber adjacent to a red fiber in the normal E D L . 95 15. High magnification longitudinal section of a white fiber in the normal E D L . 98 16. Comparative longitudinal sections of a white, intermediate and red fiber in the normal E D L . 101 17. Comparative longitudinal sections of an intermediate and red fiber in the normal soleus. 104 18. Transverse section of an intermediate fiber in the normal soleus. 106 19. Transverse section of the normal diaphragm at low and high magnifications. 109 20. Comparative transverse sections of an intermediate and a red fiber in the normal diaphragm. I l l 21. Comparative longitudinal sections of an intermediate and a red fiber in the normal diaphragm. 113 22. High magnification views of triadic couplings seen in transverse and longitudinal planes in a red fiber of the normal diaphragm. 116 23. Low magnification views of transverse and longitudinal sections of the mdx E D L . 119 24. Transverse section through portions of an intermediate fiber and a white fiber in the mdx E D L . 121 25. Myofiber degeneration in the mdx E D L . 123 26. Transverse section through a portion of a late stage degenerative fiber in the mdx E D L at low and high magnification. 125 27. Comparative longitudinal sections of an intermediate and a red fiber in the mdx soleus. 128 28. Transverse sections of the mdx soleus at low and medium magnification. 130 29. Micrograph of the mdx soleus showing several macrophages . surrounding and invading a degenerating muscle fiber. 132 x 30. Low magnification views of intermediate and red fibers of the mdx diaphragm in transverse and longitudinal section. , 135 31. Longitudinal view of a red fiber from the mdx diaphragm. 137 32. Transverse section of a regenerating fiber in the mdx diaphragm. 139 33. White fiber in the normal E D L showing examples of the two magnifications used for morphometric measurements of longitudinally-sectioned muscle fibers. 141 34. Frequency distributions of the volume fractions of mitochondria in the E D L , soleus and diaphragm in normal and mdx adult mice. 145 35. Low magnification H R S E M view of a transversely fractured portion of the E D L in the normal mouse. 170 36. High magnification transverse views comparing a white and an intermediate fiber in the normal E D L . 172 37. Comparative longitudinal views of a white and red fiber in the normal E D L . 176 38. Subsarcolemmal regions of an intermediate fiber and a red fiber in the normal E D L . 178 39. Comparative longitudinal views of an intemediate fiber and red fiber in the normal soleus. 181 40. Low and high magnification transverse views of portions of red fibers in the normal soleus. 184 41. Low magnification transverse view of the complete thickness of the normal diaphragm and higher magnification view of several muscle fibers that have been transversely fractured. 186 42. Comparative longitudinal views of an intermediate and red fiber in the normal diaphragm. 188 43. Longitudinal view showing the arrangement of mitochondria and sarcotubular elements in a white fiber of the mdx E D L . 191 44. Longitudinally fractured red fiber in the mdx E D L at low, medium and high magnifications. 193 45. Low magnification transverse and longitudinal views of muscle fibers in the mdx soleus. 196 xi 46. Longitudinal view of a red fiber i n the mdx soleus. 198 47. Examples of muscle fiber degeneration i n the mdx soleus i n transverse and longitudinal planes. 200 48. Longitudinally-fractured intermediate fiber i n the mdx diaphragm at medium and high magnification. 202 49. Red fiber i n the mdx diaphragm showing a portion of both its fiber core and subsarcolemmal region. 205 50. Longitudinally-fractured red fiber in the mdx diaphragm w i t h an extremely high mitochondrial content. 207 51. H i g h magnification longitudinal views of red fibers i n the mdx diaphragm. 209 52. Selected H R S E M stereo pairs of normal muscle fibers. 211 53. Selected H R S E M stereo pairs of mdx muscle fibers. 213 54. Effect of cerium and C a 2 + concentrations on the activity of C a 2 + -M g 2 + pNPPase i n the normal mouse gastrocnemius. 243 55. L o w and high magnification views of a white muscle fiber i n the normal mouse gastrocnemius showing the ul tracytochemical localization of C a 2 + - M g 2 + pNPPase activity. 246 56. Two recordings by x-ray microanalysis of a stained and unstained port ion of the SR membrane i n a normal muscle fiber of the mouse gastrocnemius. 248 57. Longitudinal view of a subsarcolemmal nucleus i n a white fiber of the normal gastrocnemius stained for C a 2 + - M g 2 + pNPPase. 250 58. Longi tudinal and transverse views of a red fiber i n the normal mouse gastrocnemius stained for C a 2 + - M g 2 + pNPPase. 253 59. Spectrophotometric comparison of the conversion of p N P P to pni t rophenol i n reaction media containing normal and mdx tissue. 256 60. C a 2 + - M g 2 + pNPPase reactivity i n a longitudinal section of a red muscle fiber from the mdx gastrocnemius. 258 x i i LIST O F A B B R E V I A T I O N S A N O V A analysis of variance A D P adenosine diphosphate A T P adenosine triphosphate ATPase adenosine triphosphatase °C centrigrade C a 2 + calcium ion Ca 2 + -ATPase calcium-activated adenosine triphosphatase Ca 2 + -Mg 2 + /?NPPase calcium- and magnesium-stimulated pflranitrophenylphosphatase C a C l 2 calcium chloride c D N A complementary deoxyribonucleic acid C e 3 + cerium ion C e C l 3 cerium chloride cm centimeter(s) D M D Duchenne muscular dystrophy D M S O dimethylsulphoxide EDL extensor digitorum longus E G T A ethylene glycol-bis (fi-amino-ethyl ether) N,N,N',N'-tetraacetic acid H & E hematoxylin and eosin hr hour(s) H R S E M high-resolution scanning electron microscopy/microscopic KC1 potassium chloride k D kilodalton(s) k V kilovolt(s) M molar /molar i ty mATPase myofibrillar adenosine triphosphatase m g mill igram(s) M g 2 + magnesium ion M g S 0 4 magnesium sulphate m i n minute(s) m l mill i l i ter(s) u m micrometer(s) u m 2 square micrometer(s) m m millimeter(s) m M m i l l i m o l a r x i i i m R N A messenger ribonucleic acid n number i n sample N a + sod ium N A D H - T R nicotinamide adenine dinucleotide-tetrazolium reductase n m nanometer(s) OD optical density O s 0 4 osmium tetroxide p N P P parfl-nitrophenylphosphate P 0 4 phosphate R T room temperature SD standard deviation S D H succinate dehydrogenase sec second(s) S E M scanning electron microscopy/microscopic S R sarcoplasmic reticulum T E M transmission electron microscopy/microscopic T-tubule transverse-tubule w k week(s) xiv A C K N O W L E D G E M E N T S First and foremost, I would like to extend my appreciation to my advisor Dr. Will iam Ovalle who inspired my interest and dedication to the discipline of Anatomy. I thank him for his patience, liberal guidance, persistence, and devoted interest in my education and future direction. To the other members of my committee, Dr. Darlene Reid, Dr. Mary Todd and Dr. Tim O'Connor for always being supportive and instructive during my thesis work. I would like to express an especially grateful thank you to a committee member, Dr. Wayne Vogl , who provided me with a plenitude of advice and support during my years in the Department of Anatomy. A n d special thanks to Dr. Pierre Dow not only for the support and assistance he gave me with my research, but also for his friendship. There are many other people in the department that have provided me with advice and support throughout my studies. I owe many thanks to Roseanne Mclndoe for her technical and administrative assistance over the years as well as George Spurr, Mik Tanaka and Ken Crookall for their help in the department and with animals. Many extended thanks to Drs. Harumichi Seguchi, Toshihiro Kobayashi and Teruhiko Okada at the Department of Anatomy and Cell Biology at Kochi Medical School in Japan whom I joined on a research exchange program during my Ph.D. studies. I am grateful to Dr. Elaine Humphrey at the Biosciences Electron Microscopy Facility at U B C for her assistance in electron microscopy and use of her laboratory. I am also grateful to the British Columbia Medical Services Foundation and the University of British Columbia for financial support in form of fellowships over the first three years of study and the Ministry of Education, Science, Sports and Culture of Japan who provided me with a University-to-University Cooperative Research Grant while in Japan. I would like to thank my colleagues and peers, both in and outside of the department who have made this academic and life experience enjoyable and rewarding. A n d , finally, my deepest thanks to my family who provided with heartfelt support and encouragement throughout my university education. xv G E N E R A L I N T R O D U C T I O N Overview Understanding skeletal muscle function requires a thorough comprehension of the structural design of its components. In mammalian species, skeletal muscle fibers exhibit a wide range of mechanical properties which are reflected in their composition (for reviews see Eisenberg, 1983; Gauthier, 1986; Pette and Staron, 1990). How these components are arranged in muscle fibers of functionally-diverse muscles provides important clues to understanding physiological and biochemical properties of the constituent fibers of muscles. Moreover, the structural and functional alteration of these components in disease conditions also uncovers clues to their involvement or response to disease processes which may subsequently lead to therapies in muscle disorders. Over the last century, research on skeletal muscle has included numerous approaches that span morphological, physiological, biochemical and molecular methods. A summary of relevant milestones and pertinent information on skeletal muscle structure and function is presented below. The Skeletal Muscle Fiber Skeletal muscle fibers of mammals are elongate, multinucleated cells with a cylindrical shape tapered at each end. They can range from 1 to 40 m m in length and up to 0.1 m m in diameter depending on the structure and function of a given muscle. A n entire muscle can contain from hundreds to hundreds of thousands of muscle fibers arranged in parallel that are surrounded by a sheath of relatively dense connective tissue called the epimysium. The epimysium coalesces at the end of a muscle to eventually form the tendon which then inserts into bone. The myotendinous junction is found in this region and represents the site of muscle fiber attachment to connective tissue. From the epimysium, blood vessels, nerves 1 and lymphatics enter or leave the interior of the muscle by way of fibrous partitions, termed the per imys ium, that extend into the muscle to surround bundles or 'fascicles' of muscle fibers. Furthermore, the per imysium gives rise to a delicate connective tissue, termed the endomysium, wh ich enwraps i nd iv idua l muscle fibers and supports the capillaries and nerve fibers supplying the muscle fibers. Beneath the endomysium, each muscle fiber is surrounded by both its cell membrane and an amorphous basal lamina w h i c h collectively constitute the sarcolemma. Apar t from provid ing mechanical and structural architecture, the sarcolemma also acts as a regulator of cellular attachment, migra t ion and differentiation (Schittney and Yurchenco, 1989). In the conventional light, phase contrast, or interference microscope, a longitudinal v iew of a single skeletal muscle fiber reveals a repeated and alternating pattern of dark and light bands. The dark, highly birefringent band has been termed the anisotropic (A) band, whereas the less birefringent band has been termed the isotropic (I) band. The I band is bisected by a thin dense line called the Z band (also commonly referred to as the Z line). These striations i n skeletal muscle fiber are due to the composi t ion and arrangement of a closely-packed array of small filaments, or myofilaments, into tightly packed columns called myofibrils wi th in each muscle fiber. The sarcomere is the functional unit of the skeletal muscle fiber and represents the region of the myofibril delineated by two successive Z bands. The confirmation of the filament arrangement i n skeletal muscle was largely due to the resolving power of the transmission electron microscope (TEM) upon its introduct ion to biological study. Pioneered by Hanson and H u x l e y (1953), ultrastructural observations of skeletal muscle fibers revealed that this striated or banding pattern is a consequence of two interdigitating sets of filaments, the thin and thick filaments. Thin filaments, consisting predominantly of the protein actin, and thick filaments consisting primarily of the protein myosin are arranged spatially 2 into sarcomeres, the contractile units of muscle. In mammals, thin (actin) filaments are about 1 Lim long and 8 nm in diameter and insert into the dense Z line at either end of the sarcomere. Thick (myosin) filaments are approximately 1.6 u.m long and 15 nm in diameter; they occupy the A band which is the central portion of the sarcomere (Craig, 1986). Thin filaments run between and parallel to the thick filaments and overlap with the thick filaments for some distance within the A band. As a consequence, a transverse section in the region of this filament overlap in a myofibril shows each thick filament surrounded by six thin filaments in the form of a hexagon. Based on the original sliding filament model (Huxley and Hanson, 1954; Huxley and Niedergerke, 1954), filament displacement in contraction takes place in this overlap zone and is the site where chemical energy is converted into mechanical energy (i.e. shortening of the sarcomere) by the interaction of a portion of the myosin molecule with specific sites on the actin filaments. The globular head of a myosin molecule contains an ATPase site used to energize the adjacent actin binding site by the hydrolysis of A T P . When an energized myosin binds to actin it creates an activated cross-bridge between thick and thin filaments. The highly organized structure of a muscle fiber is maintained by a cytoskeletal framework that consists of interconnecting proteins associated with membrane, contractile or soluble proteins (for review see Berthier and Blaineau, 1997). Three lattices, or domains, of the cytoskeleton in muscle fibers have been described. They include 1) the 'endosarcomeric lattice' composed of a host of proteins including the elastic titin filament and inextensible nebulin filament (Wang et al., 1985), myosin binding proteins C and H (Craig and Offer, 1976; Starr and Offer, 1983; Dennis et al., 1984) and Z line-associated a-actinin (Nave et al., 1990); 2) the 'exosarcomeric lattice' consisting of desmin- and vimentin-containing intermediate filaments (Granger and Lazarides, 1979); and 3) the 'subsarcolemmal 3 lattice' connecting the contractile apparatus to the sarcolemmal membrane and providing membrane support and integrity (Massa et al., 1994). These lattices have some proteins i n common and others that are unique, reflecting the specific structure-function requirements served by each network. More recently, the important protein composi t ion of these three lattices have been part ial ly or completely characterized and sequenced. A number of these proteins have become better understood i n recent times due to their involvement i n myodegenerative disorders, the most we l l described disorder being Duchenne muscular dystrophy (DMD) wi th its absence of the subsarcolemmal protein, dystrophin (Hoffman et al., 1987). Our knowledge of the array of cytoskeletal proteins i n muscles has expanded wi th new advances i n molecular techniques and it has become more apparent that the ability of a muscle fiber to establish, stabilize and maintain its architecture depends on cytoskeletal integrity. Internal Membrane Systems of Skeletal Muscle Fibers In addition to closely packed myofibrils and cytoskeletal components, variable numbers of mitochondria and an elaborate network of membranous tubules and cisternae, constituting the sarcotubular system, also occupy the sarcoplasm of the muscle fiber. Mitochondria are aligned i n strategic sites w i t h i n the muscle cell, usually at the level of the I band and also in aggregates at the periphery of the fibers (Gauthier, 1969). They represent the site of oxidative phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) and, thus, provide the necessary energetics for muscle contraction and maintenance of muscle fiber homeostasis (Lieber, 1992). The fine structural complexity of mitochondria i n skeletal muscle fibers of mammals has become increasingly evident since their in i t ia l observations and descriptions i n early T E M studies (Padykula and Gauthier 1967; Gauthier, 1969; 4 Schiaffino et al., 1970). Variations i n shape and size of these organelles are often not appreciated i n two-dimensional images w i t h conventional T E M thin sectioning methods, and, as a result, textbook images have commonly portrayed them as discrete, spherical or sausage-shaped organelles situated between the myofibrils (Cormack, 1987; Fawcett, 1994). However, a much more elaborate configuration of mitochondria has been noted i n mammal ian skeletal muscle fibers. These organelles have been described as being organized into a framework of two grids relative to the myofibril lar contractile apparatus (Kamieniecka and Schmalbruch, 1980). In longitudinal sections, mitochondria that are oriented i n the tranverse grid relative to the axis of the fiber appear as thin circular profiles on both sides of the Z bands. These transversely-oriented pairs of mi tochondr ia par t ia l ly encircle myofibrils at the level of the I band and can exhibit branches that course between neighbouring myofibri ls at the I band level of the sarcomere. In addit ion to mitochondria oriented i n the transverse plane, mi tochondr ia l - r ich fibers may contain a longi tudinal ly-or iented mitochondria l network that forms columns paral lel to the myofibr i ls w h i c h interconnect w i t h the transverse grids of mi tochondr ia . Some researchers have postulated that these two grids of mitochondria form a three-dimensional in ter l inking network throughout the entire length of some skeletal muscle fibers (Bubenzer, 1966; Bakeeva et a l , 1978). Another spatial inhomogeneity i n the distr ibution of mitochondria that occurs primarily i n highly oxidative skeletal muscle fibers is the accumulation of spherical-to -ovo id mi tochondr i a under the sarcolemma (Romanu l , 1965). These subsarcolemmal clusters of mitochondria are often loca l ized to sites where capillaries come into close contact w i t h the muscle fiber surface and have been proposed to represent the regions where the biochemical exchanges between the blood and the muscle fiber take place (Romanul, 1965). 5 The sarcotubular system encircles each myof ib r i l and comprises the transverse (T) tubules and sarcoplasmic reticulum (SR) which together play a critical role i n excitation-contraction coupling (Sandow, 1965; Caputo, 1978). T-tubules are thin invaginations of the cell membrane found at the A - I junction i n mammalian muscle fibers that course transversely across the muscle fiber and function to transmit an action potential deep into the fiber. The SR, on the other hand, is a closed membranous system of anastomosing tubules wi th in the muscle fiber that functions i n the storage, release and sequestering of C a 2 + by a series of channels and pumps embedded wi th in its membrane (Martonosi, 1986). These two systems ensheath the myofibrils and form triadic or diadic couplings opposite the A - I band junction i n mammalian skeletal muscle fibers (Gauthier, 1969). Configurations of the SR i n a l l types of muscle fibers are formed by a combination of fenestrated, tubular and junctional regions (Franzini-Armstrong, 1986). The sheath of SR immediately surrounding the A band has been termed A band SR whereas those portions of the SR in the I band are termed I band SR (Porter and Palade, 1957). A t the central region of the A band SR, small to large fenestrations are typ ica l ly observed. These are formed by an extensive anastomosing network of the SR tubules. Because of its appearance and circumscription around a myofibril it is commonly referred to as a fenestrated collar (Franzini-Armstrong, 1986). Extending from the fenestrated collar are straight tubular regions that course towards the A - I junction where they form a cisterna adjacent to the T-tubules. This cisterna has been referred to as either the terminal cisterna, the lateral sac, or as the junctional SR (Franzini-Armstrong, 1986). In this thesis, terminal cisterna(e) w i l l be used to describe the port ion of the SR that is found adjacent to the T-tubule at the triad. The I band SR is variable but typically assumes a three-dimensional network of anastomosing tubules. It also shows an intimate association wi th the I band mitochondria. Finally, Go lg i complexes, rough 6 endoplasmic reticulum and lysosomes are only rarely observed in normal adult muscle fibers, but are usually located adjacent to the nuclei at the periphery of the muscle fiber. Sequence of Events in Excitation-Contraction Coupling and Relaxation Excitation-contraction (E-C) coupling in skeletal muscle fibers can be divided into a sequence of events that begin with a nervous impulse and end with shortening of the muscle cell. Following the contraction process, relaxation of the fiber returns it to its original state and restores its ability to contract again. The steps involved in E - C coupling and relaxation of various types of muscle fibers have become better understood over the past several decades and numerous reviews of this process have been published (Sandow, 1965; Schmidt, 1978; Horowicz, 1986; Martonosi, 1986). Initiation of this sequence begins with the arrival of an action potential at the synaptic terminal of a motor neuron at the specialized region of the muscle fiber surface known as the neuromuscular junction (NMJ). Depolarization of the motor neuron membrane at the presynaptic terminal results in rapid release of the pre-packaged neurotransmitter, acetylcholine, from small vesicles within the nerve terminal into a narrow, intervening space between the nerve terminal and muscle fiber termed the synaptic cleft. Once released into the synaptic cleft, acetylcholine diffuses across the narrow space and binds to acetylcholine receptors localized in the postsynaptic membrane of the muscle fiber. The postsynaptic membrane of muscle fibers is commonly referred to as the 'motor end plate' and is typified by undulating folds that serve to increase the surface area of the muscle membrane at the N M J (Padykula and Gauthier, 1970). Subsequent binding of acetylcholine to their receptors on the postsynaptic membrane results in an increase in permeability of the endplate membrane to sodium and potassium ions (Magleby, 1986). The influx of sodium and efflux of potassium through acetylcholine-activated channels leads to a 7 depolarization of the motor endplate membrane generating a so-called 'endplate potential'. The endplate potential causes voltage-gated ion channels in the surrounding sarcolemma to open, intiating an all-or-none action potential along the muscle membrane. In vertebrate skeletal muscle fibers, the action potential is propagated not only along the entire length of the muscle fiber, but also in the radial direction along the regularly arranged system of narrow T-tubules which are continuous with the sarcolemma (Huxley and Taylor, 1958; Sandow, 1965; Smith, 1966; Franzini-Armstrong, 1973; Caputo, 1978). T-tubules, therefore, are important for a widespread and efficient delivery of the signal to inner aspects of the muscle fiber resulting in a more even contraction of the muscle fiber as a whole. Along the length of T-tubules, specialized contacts are made with the SR at sites called triads or diads which consist of a central T-tubule and either one or two associated terminal cisternae of the SR (Sandow, 1965; Smith, 1966). In mammalian skeletal muscle fibers, these contact sites are localized at the A-I junctions of the sarcomere. The signal for excitation-contraction coupling passes from the T-tubule to the SR at triads to stimulate the release of C a 2 + from the terminal cisternae of the SR. Ultrastructural observations have revealed a unique structural linkage between the T-tubule and the SR membranes in the form of periodic electron densities that span the gap between these two membrane systems; these were first described at the triad in T E M studies of frog twitch skeletal muscle by Franzini-Armstrong (1970) and have been termed 'junctional feet'. Embedded within the SR terminal cisternae are C a 2 + channels that open in response to the depolarization of the T-tubule membrane. This transfer of signal to the SR has recently become attributed to two receptors; the dihydropyridine receptor associated with the T tubule membrane and the ryanodine receptor associated with the junctional face of the terminal cisternae (for review see Franzini-Armstrong and Protasi, 1997). It has been postulated that the depolarization of the T-tubule membrane causes a charge movement in the 8 voltage sensor of the dihydropyridine receptor that stimulates a conformational change in the ryanodine receptor which, in turn, opens its associated C a 2 + channel. The coupling of the dihydropyridine receptor and ryanodine receptor is believed to be mediated through a recently discovered integral membrane protein of the SR called 'triadin' which occurs as an abundant 90 kD membrane protein co-localized with the C a 2 + release channel on the terminal cisternae of the SR (Caswell et al., 1991; Guo et al., 1994). Its localization to terminal cisternae of the sarcoplasmic reticulum suggests that it has an important role in excitation-contraction coupling (Guo et al., 1994). The C a 2 + released by the opening of the ryanodine channel in the SR causes a dramatic increase in sarcoplasmic C a 2 + concentrations which rise from approximately 0.1 u M to 10 u M at the height of contraction (Perry, 1986). Once C a 2 + is released from the SR, it diffuses into the vicinity of the myofilaments and binds to troponin, a part of the contractile protein regulatory apparatus localized on the thin filament (Ruegg, 1988). When C a 2 + binds to troponin (C), a structural change occurs in the troponin molecule and the interaction between troponin and the actin-binding protein, tropomyosin, is altered in such a way that tropomyosin uncovers the myosin binding site on the actin filament. The myosin molecule contains a globular head that has an ATPase site used to energize the adjacent actin-binding site by the hydrolysis of A T P (Craig, 1986). The energized myosin will then bind actin, creating an activated cross-bridge between the thick and thin filaments. Formation of the actomyosin complex results in the release of inorganic phosphate from the complex and the energy stored in the cross-bridge is used to propel the thin filament past the thick filament resulting in the shortening of the sarcomere and the generation of force. After cross-bridge activation, the spent A D P must be released and another molecule of A T P attached to the actomyosin complex in order for the two filaments to dissociate. This process of attachment, release and 9 reattachment of the actin and myosin molecules will continue as long as free C a 2 + levels are maintained at high (10 uM) levels (Huxley, 1969). Relaxation of muscle is largely dependent on the active removal of C a 2 + from the regions near the myofilaments back into the lumen of the SR. This is initiated by polarization of the surface membrane and T-tubules and the closing of the C a 2 + channels (Horowicz, 1986). Removal of the free cytosolic C a 2 + is accomplished predominantly by a highly efficient C a 2 + and M g 2 + dependent ATPase pump embedded within the SR membrane that couples the hydrolysis of 1 A T P to the translocation of 2 C a 2 + from the cytoplasmic regions to the lumen of the SR (Martonosi, 1986). A more complete description of the Ca 2 + -pumping mechanism in the SR is presented in Chapter 4 of this thesis. In addition to the rapid uptake of C a 2 + by the SR pump, a high capacity, low affinity, intraluminal Ca 2 + -binding protein named calsequestrin provides a Ca 2 + -buffering mechanism that is believed to substantially increase the C a 2 + storage ability of the SR (Martonosi, 1986). Skeletal Muscle Fiber Types It was first recognized over a century ago that the colour of femoral muscles of the rabbit could be related to differences in speed of contraction; red muscles were typically slow contracting whereas white muscles were fast contracting (Ranvier, 1873). In the time following this initial observation, extensive research into mammalian skeletal muscle fiber diversity has been undertaken. Skeletal muscle fibers can now be classified more precisely according to innervation patterns (Burke, 1981), contractile properties (Burke et al., 1973), histochemical staining patterns (Ogata, 1958a-c; Engel, 1962; Brooke and Kaiser, 1970; Pette and Staron, 1990), ultrastructural features (Padykula and Gauthier, 1967; Eisenberg and Kuda, 1976), or by immunocytochemical staining properties (Gauthier and Lowey, 1979; Pette and Staron, 1990). 10 Within one skeletal muscle of an animal there typically exists a mixture of fibers characteristic to that muscle. This can show variability between species and strains and reflects the heterogeneity of this tissue, its functional specialization and subsequent functional plasticity (for review see Pette and Staron, 1997). Of the numerous techniques used for distinction of fiber types, none have been more commonly applied than those based on histochemical methods. In a mixed population of muscle fibers, fiber types can be classified according to mitochondrial content by routine histochemical staining for succinate dehydrogenase (SDH), cytochrome oxidase, or nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) (Ogata, 1958a-c; Novikoff et al., 1961). Muscle fibers with abundant mitochondrial enzyme activity have been called 'red' fibers. Conversely, muscle fibers with low activity have been termed 'white' fibers. A n 'intermediate' fiber has also been described which shows moderate mitochondrial enzyme activity. Differences between these three main fiber types, therefore, relates primarily to the aerobic potential associated with each fiber (Hoppeler, 1986). Futhermore, this terminology has also been applied to electron microscopic studies because identification of mitochondria allows the same fiber type to be observed at both the light and electron microscopic level (Gauthier, 1969). Another widely used classification scheme that was originally developed by Engel (1962) is the histochemical staining for myofibrillar adenosine triphosphatase (mATPase). It was found that muscle fibers could be classified into two categories depending on whether they possessed low (type I) or high (type II) mATPase activity under alkaline conditions (Engel, 1962). Following acid preincubations, a reversal in staining is generally observed indicating that the activity of mATPase in different muscle fibers shows sensitivity to the p H of its surroundings. In 1970, Brooke and Kaiser developed a staining method in which the type II fibers were further subdivided into type Ha and type lib based on sensitivity of mATPase activity to 11 variations in p H levels in the acidic range. At p H 4.3, both type Ila and type lib fibers are inhibited, but at p H 4.5 the type lib fiber shows moderate activity. A further identified subtype known as the type lie fiber is thought to represent a transitional fiber type since it exhibits staining throughout a wide p H range used for mATPase fiber typing, although it is usually found in small proportions in muscle (Brooke and Kaiser, 1970). Differences in mATPase staining have also been correlated to myosin composition by the response of the two categories of fibers to immunocytochemical labelling with antibodies to myosin heavy chain (MHC) isoforms (Gauthier and Lowey, 1979; Pette and Staron, 1997). The number of M H C isoforms continues to increase as methods become more refined and are applied to an increasing number of different muscles (Pette and Staron, 1997). Some common M H C isoforms include those that correspond to histochemically-identified (mATPase) fiber types. MHCIIb, MHCIId /x , MHCIIa and M H C I are myosin isoforms encountered in adult limb and trunk musculature of small mammals (Pette and Staron, 1997). Fibers labelled with antibody for fast myosin (MHCIIb, MHCIIa) display high mATPase activity under alkaline conditions and low activity under acid conditions; these are most often found in fast-twitch motor units (Burke, 1981) and correspond to the type II fibers. These fibers typically stain darkly for glycolytic enzymes and can exhibit a wide range of staining intensities for oxidative enzymes. Slow myosin-labelled (MHCI) fibers correspond to type I fibers and stain lightly for mATPase after alkaline preincubation and dark for mATPase after acid preincubation; they are usually found in motor units with slower contraction times (Burke, 1981). These fibers stain lightly for glycolytic enzymes but darkly for oxidative enzymes. O n the basis of glycogen content and oxidative enzyme capacity, a nomenclature has been devised that combines metabolic enzyme-based properties and mATPase-based properties, as well as fast and slow M H C immunolabelling. Using combinations of these parameters, 12 the distinction of three fiber types termed slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), and fast-twitch glycolytic (FG) have been proposed (Barnard et al., 1971; Peter et al., 1972; Anderson et al., 1988). The fiber type composition of a particular muscle can exhibit considerable variability in neuromuscular disorders such as the muscular dystrophies and motor neuron diseases (Banker and Engel, 1986). Application of morphological and histochemical techniques to the study of muscle-wasting diseases has become an important area of biomedical research and these techniques are essential for the determination of the functional status of skeletal muscle in health and disease. The next section serves as an introduction to neuromuscular disease as it relates to D M D and animal models used for its study. Duchenne Muscular Dystrophy, the Mdx Mouse, and Other Animal Models Muscular dystrophy is a term used to describe the heterogeneous group of genetically-linked disorders that cause progressive weakness and wasting of the skeletal muscles. The most common and devastating of these disorders in humans is the X-linked recessive Duchenne muscular dystrophy (DMD) which was first described in the mid-1800's by Meryon (1852) and later by Duchenne (1868). The incidence of this disease in North America has been estimated to be one in 3500 boys, and in approximately one-third of cases the disease is caused by a spontaneous mutation in a gene located on the Xp21 chromosome in D M D patients. In the remaining two-thirds of cases the defective gene is inherited on the X chromosome from a carrier mother (Worton, 1992). In humans, this genetic disorder exhibits no obvious clinical manifestation until the age of three to five years, when proximal muscle weakness is first observed. The ensuing progressive loss of muscle strength usually leaves affected individuals wheelchair-bound by the age of 11, and results in early death due to 13 respiratory failure. To date there is no cure and no effective treatment although prednisone, a catabolic steroid, has, paradoxically, been shown to stabilize muscle strength for a period of up to three years (Brooke et al., 1989). The Becker type of muscular dystrophy (Becker and Kiener, 1955) is a milder form of the disease w i t h an incidence rate of about one-tenth of that of the severe Duchenne form (Becker, 1964). It is characterized by a much later onset w i th loss of ambulation after the age of 16 years and a relatively normal lifespan (Becker, 1964). Although thought for years to be a distinct disease, it is now recognized to be caused by mutations i n the same gene, and therefore a milder version of the same disease (Baumbach et al., 1989; Koenig et a l , 1989). The basis of these disorders stemmed from molecular genetic studies using a strategy based on identification of sequences that mapped into a region of the X chromosome. This region was previously shown by Kunke l and colleagues (1985) to be deleted i n a boy wi th several X-l inked disorders, including Duchenne muscular dystrophy. The identification of a clone from this part of the X chromosome that detected deletions i n 6-7% of Duchenne patients suggested that this was the site of the Duchenne gene. Conserved sequences from this region of the X chromosome were used to identify c D N A (complementary D N A ) clones made by reverse-transcribing m R N A (messenger R N A ) . The sequences encoded i n the c D N A were shown to be deleted i n a number of boys wi th D M D . This provided evidence that the isolated c D N A clones were indeed from the gene that is mutated i n muscular dystrophy. The gene, known as the D M D gene, was shown to encode a protein that Hoffman and Kunkel 's laboratory (Hoffman et al., 1987) named dystrophin, and as expected, this protein was missing from the skeletal muscle tissue of boys wi th D M D (Monaco et a l , 1986). Once the c D N A had been isolated and sequenced, the deduced amino acid sequence of dystrophin appeared to resemble that of other cytoskeletal proteins such 14 as alpha-actinin and spectrin, suggesting that dystrophin is itself a cytoskeletal protein (Koenig et a l , 1988). Dystrophin has been shown to be associated with a complex of sarcolemmal glycoproteins that are believed to provide a linkage to the extracellular matrix protein, laminin (Campbell and Kahl, 1989; Ervasti et al., 1990; Ohlendieck et al., 1991; Ibraghimov-Beskrovnaya et al., 1992). The absence of dystrophin leads to a dramatic reduction in these dystrophin-associated glycoproteins in the sarcolemma of patients with D M D and mdx mice (Ervasti et al., 1990; Ohlendieck et al., 1991). Furthermore, it has been demonstrated that a dystrophin-related protein named utrophin (Matsumura et al., 1992; Tinsley et al., 1992), an autosomal homologue of dystrophin, is associated with an identical or antigenically similar complex of sarcolemmal proteins in skeletal and cardiac muscle of both normal and mdx mice. Metabolic defects have also been proposed as a possible contribution to myofiber destruction in D M D . Some researchers have proposed that mitochondrial calcium overloading may represent a general mechanism for cell necrosis in disease and the overloading and subsequent release of stored calcium may be sufficient to promote the rapid degeneration of cellular components (Wrogemann and Pena, 1976). Although numerous theories for myofiber destruction in this disorder have been proposed, the precise mechanism remains obscure. The mdx mutant of the C57BL/10SnSn mouse strain has been utilized in the past 15 years as an animal model applicable to the study of X-linked human muscular dystrophies (Bulfield et al., 1984). In several of the earliest pathological studies of the mdx mouse, it was recognized that some of the histopathological features resembled quite closely those seen in D M D (Bulfield et al., 1984; Anderson et al., 1987,1988; Carnwath and Shotton, 1987; Torres et a l , 1987; Coulton et al., 1988; Cullen and Jaros, 1988) while others did not fit at all well (Anderson et al., 1988). More importantly, however, was the fact that the same biochemical defect was 15 shown to be present in mdx skeletal muscle, in that it lacked the protein dystrophin (Hoffman et al., 1987). Analysis of the dystrophin gene in the mdx mouse revealed that the genetic defect was a point mutation involving a single base change within an exon (Sicinski et al., 1989). This feature of the mdx mouse made it a reliable model for characterizing the primary pathological processes linking the absence of dystrophin to necrosis in skeletal muscle fibers. The high regenerative capacity in muscles of the mdx mouse, however, is a unique feature of this animal model that sets it apart from the human D M D condition (Anderson et al., 1988). This has been noted to be especially true for the slow-twitch soleus and to a lesser extent in the fast-twitch E D L (Anderson et al., 1988). More recently, however, the diaphragm of adult mdx mice has been shown to contain a pattern of degeneration and fibrosis similar to that of D M D limb muscles (Stedman et al., 1991; Dupont-Versteegden and McCarter, 1992). It has been proposed that the continuous activity of the diaphragm coupled with a low threshold for work-induced injury (Weller et al., 1990; Stedman et al., 1991; Dick and Vrbova, 1993) may make this muscle more susceptible to progressive degeneration. Dystrophin has also been shown to be missing in two other animals, the X M D dog and the dystrophic cat (Hoffman et a l , 1987; Cooper et al., 1988; Carpenter et al, 1989), although experimental research on these animals has been fairly limited due to their size and cost of maintenance. The short breeding time and relative cost and availability of the mdx mouse has made it the model of choice for studies on the pathophysiology of D M D . Other mouse models for progressive muscular dystrophy have also been described - the 129/ReJ d y / d y (Michelson et al., 1955) and the C57BL/6J d y 2 J / d y 2 J (Meier and Southard, 1970). Muscular dystrophy in these mouse models is a result of an autosomal recessive gene defect (Harris and Slater, 1980) that results in extensive muscular degeneration. Skeletal muscles of the d y / d y mouse model have been found to be deficient in the basement membrane protein merosin 16 (Sunada et al., 1994; Nonaka, 1998). These animal models have typically been identified because of demonstration of similar defects in specific proteins or genes with humans and, in some cases, these defects correspond to proteins linking the subsarcolemmal cytoskeleton to the extracellular matrix. Since the mdx mouse has become an important animal model for myopathic research and because it shares a similar genetic mutation with D M D in humans, it would be useful to clarify some of the histochemical and ultrastructural features of its muscle fibers. To date, there are few studies on the structure and function of the internal membrane systems of mdx muscle fibers (Cullen and Jaros, 1988; Kargacin and Kargacin, 1996; Khammari et al., 1998; Kuznetsov et al., 1998). Moreover, two of these groups have reported contradictory results with respect to the function of the SR in mdx muscle fibers (Kargacin and Kargacin, 1996; Khammari et al., 1998). It is predicted that the underlying defects in mdx muscle pathology and its ability to efficiently regenerate may be a result of modifications of the internal membranous structures of the muscle fibers. Statement of Objectives and Hypotheses This project has been divided into four chapters that deal with specific questions on the structure and function of the internal membrane systems of normal and mdx-dystrophic skeletal muscle fibers. The main purpose of the first chapter was to describe by light microscopy the morphological and histochemical differences that exist within three functionally-diverse muscles of the adult mouse in normal and mdx-dystrophic conditions. I wished to test the hypothesis that the oxidative capacity of mdx-dystrophic muscles is altered relative to their normal counterparts. Mitochondrial activity, detected by the N A D H - T R stain, was analyzed by quantitative histochemical methods to compare muscle fiber types of the E D L , soleus and diaphragm with those in the mdx mouse. At the ultrastructural level, 17 conventional T E M is utilized in the second chapter to test the hypothesis that mitochondria and the sarcotubular system are distributed differently in fiber types in these three muscles and are altered in dystrophic muscle. The third chapter employs state-of-the-art high-resolution scanning electron microscopy (HRSEM) to provide insights into the three-dimensional ultrastructure of mitochondria and the sarcotubular system in murine muscle fibers. This methodology will be used to test the hypothesis that functional diversity in fiber types of normal and mdx muscle is reflected in the spatial orientation and distribution of these organelle systems. Finally, the fourth chapter was undertaken to test the hypothesis that Ca 2 + -pumping mechanisms within the SR are altered in muscle fibers in the mdx condition. Alterations in this activity may be responsible for the disturbance in the C a 2 + homeostasis of dystrophic muscle. 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I N T R O D U C T I O N Fiber type differences in mammalian skeletal muscles have been elucidated using morphological, histochemical, immunohistochemical, biochemical and physiological criteria (for reviews see Gauthier, 1986; Pette and Staron, 1990, 1997; Hamalainen and Pette, 1995). Information from each of these parameters has led to a wide range of fiber type nomenclatures that evolved to suit the chosen methodologies (Gauthier, 1986). One particular classification scheme proposed for mammalian muscle fiber types is based on the oxidative enzyme capacity within a given muscle fiber (Ogata, 1958; 1964; Gauthier and Padykula, 1966). Staining at the light microscopic level for mitochondrial-limited oxidative enzymes such as succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide reductase (NADH) has revealed three major types of fibers based on their staining intensities and relative mitochondrial content. Muscle fibers rich in mitochondria have been termed 'red' fibers, since they were observed by early microscopists to predominate in red muscles of mammals (Ranvier, 1873). In contrast, fibers with a paucity of mitochondria have been designated 'white' fibers, originally named for their predominance in white muscles. Fibers with a moderately high mitochondrial content and intermediate between those of 'red' and 'white' fibers have been termed 'intermediate' fibers. Within a given muscle, there usually exists a 'mosaic' of these fiber types that relate to the structural and functional properties of the muscle as a whole (Gauthier, 1986). 27 Comparative measurements of oxidative enzyme activity in single skeletal muscle fibers can be obtained with a high degree of accuracy by microphotometric analysis of reaction product produced by SDH or NADH-tetrazolium reductase (NADH-TR) stains (Pette et al., 1979; Sieck et al., 1986). These quantitative histochemical methods have also been used in combination with myosin adenosine triphosphatase (mATPase) fiber typing to correlate oxidative capacity to type I, type Ha and type lib fiber types (Brooke and Kaiser, 1970) in studies on the mammalian diaphragm (Green et al., 1984; Reid et al., 1992; Sieck et al., 1995). These studies have indicated a wide range of oxidative enzyme activities in fiber types of a given muscle (Sieck et al., 1986; Reid et al., 1992) and between the corresponding muscle fiber types of different species (Green et al., 1984). Alterations in the normal oxidative capacity of skeletal muscle fibers have been reported in a number of neuromuscular diseases (for review see DiMauro et al., 1998). In human Duchenne muscular dystrophy (DMD), it has been postulated that either defects in the ATP pathway (Bonsett and Rudman, 1992) or Ca 2 + overloading of mitochondria (Wrogemann and Pena, 1976) may contribute to the progressive muscle fiber necrosis seen in this disease as well as in other dystrophin-defective muscular dystrophies. While several animal models currently exist for research on human D M D including the X M D dog and the dystrophic cat (Hoffman et al., 1987; Cooper et a l , 1988; Carpenter et al., 1989), none have been more widely employed in recent times than the mdx mouse (Bulfield et a l , 1984). The mdx mutant mouse was originally identified from an inbred C57BL/10ScSn strain by Bulfield and coworkers (1984) as a possible model for muscular dystrophy by detection of elevated serum pyruvate kinase levels and skeletal muscle histopathology of limb tissues. Subsequent studies on the mdx mouse have revealed that it shares a common X-linked recessive genetic defect with that of human D M D (Sicinski et al., 1989). Like skeletal muscles of D M D patients, 28 the skeletal muscle fibers of mdx mice also lack the subsarcolemmal protein, dystrophin (Hoffman et al., 1987). Why dystrophin's absence leads to deleterious consequences in skeletal muscle tissues, however, still remains unsolved. Studies pertaining to the mitochondria and oxidative capacities of mdx mouse skeletal muscle fibers are relatively few in number (Glesby et al., 1988; Dunn et al., 1993; Even et al., 1994; Kuznetsov et al., 1998). In a recent study on the mdx quadriceps muscle (Kuznetsov et al., 1998), the oxidative capacity of skeletal muscle fibers was shown to be reduced by up to 50% in comparison to normal muscle. From these data, it has been postulated that a disruption in energy metabolism in mdx muscle tissue may be associated with the myodegenerative process observed in this disease. It has not been determined, however, whether oxidative differences exist in functionally diverse skeletal muscles or whether they are limited to specific fiber types within a given muscle. To date, there are no quantitative histochemical studies on the oxidative enzyme capacities between normal and mdx muscle tissue, especially those pertaining to functionally distinct muscles and specific muscle fiber types. This study was undertaken, therefore, to test the hypothesis that the oxidative capacity of functionally diverse skeletal muscles of mdx mice is altered as a result of the disorder, and that this difference is related to changes in specific muscle fiber types of a given muscle. Oxidative capacities of individual fibers were examined by quantitative microphotometric techniques applied to N A D H - T R stained cryosections of the E D L , soleus and diaphragm muscles of adult mdx mice and they were compared to those of their normal age-matched counterparts. 29 M A T E R I A L S A N D M E T H O D S Animals and Tissue Preparation Adul t mice (25-32 wk) of the normal C57Bl/10ScSn (n=5) and mdx C57Bl/10ScSnMdx (n=5) strains were used in this study. Three different skeletal muscles were examined; two hindlimb muscles, the extensor digitorum longus (EDL) and soleus, and the diaphragm. These muscles were chosen because, in the C57BL/10ScSn control strain, each muscle exhibits distinct functional, morphological and histochemical features that have been documented extensively by other investigators (Bressler et al., 1983; Ovalle et al., 1983; Green et al., 1984; Anderson et al., 1987, 1988; Stedman et al., 1991). Moreover, the diaphragm of adult mdx mice has been shown to exhibit a pattern of muscle fiber degeneration and fibrosis similar to that reported in human D M D limb muscles (Stedman et a l , 1991; Dupont-Versteegden and McCarter, 1992; Petrof et al., 1993). Normal and mdx mice were killed simultaneously with an overdose of chloroform. The E D L , soleus and diaphragm muscles were rapidly excised and kept moist with a physiological saline-soaked gauze pad. To facilitate the comparison and speed of preparation between specimens, normal and mdx muscles were oriented side-by-side between two thin slices of calf liver and quickly frozen by plunge immersion in isopentane cooled to -196°C with liquid nitrogen. Samples were then transferred to a Bright Instruments 5030 series cryostat microtome (Huntington, England) and allowed to equilibrate to - 1 8 ° C . They were then mounted for transverse sectioning on a cryostat chuck with O . C . T . embedding medium (Miles Laboratories, Naperville, IL). Serial cryosections (8 um thick) from midbelly portions of the E D L and soleus, and from the costal region of the diaphragm were cut with a metal knife and collected on 22 m m 2 coverslips. Since maintenance of section thickness was of importance in this study, all samples were 30 cut the same day and without adjustment of the settings on the cryostat (i.e. thickness control was not altered). Variation in thickness between sections of normal and mdx muscles was minimized by examination of side-by-side orientations of muscle samples within the same tissue section. NADH-TR Staining The N A D H - T R histochemical reaction of Novikoff et al. (1961) was used to determine relative levels of oxidative enzyme activity associated with mitochondria of muscle fibers in frozen cross-sections. Sections were stained at room temperature in a Tris-buffered (0.1 M) solution containing 1 m M of fi-nicotinamide adenine dinucleotide, reduced form ( N A D H ) , and 1 m M nitro-blue tetrazolium as an electron acceptor (Sigma, St. Louis, MO). After performing various incubation times for N A D H - T R , 30 min was determined as the optimal time for considerable fiber variability in staining. After incubation, the coverslips were rinsed well in five changes of distilled water and then dehydrated through an ascending series of ethanols (70, 80, 90, 100%) for two minutes each, cleared in xylene and mounted with cytoseal on glass slides. Standardization of N A D H - T R staining was attained by sectioning and staining all samples within a 24 hr time period. A l l muscle samples (n=5 for each muscle) were stained simultaneously with the same stock staining solution. Myofibrillar ATPase Staining A modified method for myofibrillar ATPase (mATPase) detection based on the use of metachromatic dyes was employed to determine the presence of type I, type Ha and type lib fiber types in muscle samples (Doriguzzi et al., 1983). This method is based on phosphate content after incubation in the ATP-containing reaction medium and is associated with acid stability of myofibrillar ATPase activity 31 within fiber types. This mATPase method was adopted because it is less time consuming than previously published protocols and is relatively safe since the ammonium sulfide step is eliminated (Johnson and Ovalle, 1986). Moreover, at p H 4.5, three extrafusal fiber types are simultaneously revealed - type I fibers are dark staining, type Ha fibers are pale staining, and type lib are intermediate in their staining intensity (Brooke and Kaiser, 1970; Doriguzzi et al., 1983). Sections were preincubated for 30 sec to 3 min in an acidic medium containing 100 m M sodium acetate buffer with 100 m M potassium chloride at p H 4.5, washed twice for 60 sec in basic medium containing 50 m M glycine buffer and 100 m M sodium chloride (pH 9.4), and then incubated in a 3 m M adenosine triphosphate (ATP) basic medium (pH 9.4) at 37°C for 30 min. Following incubation, the sections were stained with a 1% aqueous azure A solution for 10 sec and then rinsed with 5 changes of distilled water. Sections were then dehydrated rapidly in ascending ethanols (70, 80, 90, 100%), cleared in xylene and mounted with cytoseal on glass slides. Sections stained with Azure A displayed a clear cut differentiation of muscle fibers which could be distinguished by their relative intensities to one another. Type I fibers stained darkly following acid preincubation whereas type Ha fibers stained lightly. Type lib fibers, on the other hand, possessed an intermediate staining intensity. Preliminary staining experiments indicated that timing of the acidic medium preincubation step varied for all three muscles to attain good fiber type distinction. The major criterion for the selection of appropriate preincubation condition was that there had to be a clear pattern of relative stain intensity between the different fiber types in each muscle. Inhibition of type Ha fibers in the E D L was the most time-sensitive and showed optimal distinction of fiber types at 30-45 sec preincubation times at p H 4.5. Optimal distinction of the three fiber types in the diaphragm was 90 sec preincubation, whereas that for the soleus was 3 min in the acid preincubation medium. 32 Serial sections were also stained with hematoxylin and eosin (H&E) to assess the morphology of the samples and to determine the presence of central nucleation in muscle fibers as well as myonecrotic areas. Following H & E staining, the coverslips were rinsed in ascending ethanols (70, 80, 90, 100%) for 2 min each, cleared in xylene, and mounted in Cytoseal 280 (Stephens Scientific, NJ). Photographs were taken on a Leitz Orthoplan photomicroscope using T-Max 100 Black and White Kodak film. Quantitation and Image Analysis of NADH-TR-Stained Sections Since the primary objective of this study was to quantitate histochemical signals, the parameter measured was optical density (OD) of the reaction product in cryostat sections. Serially-sectioned muscle fibers were initially matched and numbered under light microscopy following staining with H & E , N A D H - T R , and mATPase (pH 4.5). Corresponding sections of each stain were then captured with a C C D Spot camera mounted on a Zeiss brightfield microscope. Digitized images of all samples were obtained in one session to ensure consistent lighting and microscope settings. Images were obtained with a x20 objective and captured in the green channel as a 1.2 M B 8 bit mono tiff file at a resolution of 1315 x 1033 pixels. To calibrate the system to the specific needs of this study, camera levels were calibrated to a dark reference and light reference and a grey level scale set to 0 and 255 respectively. Grey scale measurements were then automatically converted to optical density units using the equation O D = loglO (255/255 - x) where x equals the averaged gray scale value from selected areas. From this equation a value in the range of 0 - 2.71 is obtained where 0 is equivalent to a black background (no light to camera) and 2.71 corresponds to a white background (coverslip, mounting medium and glass slide). The image analysis system was also calibrated for morphometry 33 with a stage micrometer. By using a x20 objective, each pixel had a projected area of 0.35 um 2 . Profiles of 25 neighbouring muscle fibers from 5 regions of each muscle section were randomly chosen and analyzed with a web-based analytical software program, Scion Image (v. 1.62) to measure fiber areas (in u.m2) and their relative optical densities (in O D units). The boundaries of 25 muscle fibers were outlined on each digitized image and subsequent measurements then transferred to an Excel file. To avoid the influence of any regional differences in areas or N A D H - T R activity, muscle fibers were selected from 5 representative areas across the sections of the E D L and soleus, and equally from the abdominal and thoracic sides of the diaphragm. A n example of the sampling method for one muscle is shown below: Section Field Fibers EDL 1 25 2 25 3 25 4 25 5 25 125 fibers/muscle Care was taken to completely encircle muscle fibers with the tracing tool so as to include the whole muscle fiber including the dense subsarcolemmal staining in some of the fibers. The O D for all the pixels within the outlined fiber was averaged by the software to determine the mean N A D H - T R staining within the cross-sectional area of the fiber. This averaging procedure reduced possible errors resulting from areas of higher N A D H - T R staining density within whole muscle fiber cross-sections, eg. higher subsarcolemmal N A D H - T R staining density. A fiber core measurement was also obtained by tracing a region within the fiber's boundaries to approximately 50% of its volume. This removed the dense peripheral 34 rim of mitochondria typically observed in some mitochondria-rich fibers and allowed possible differences in subsarcolemmal mitochondrial activity to be detected. Centrally-placed nuclei were also avoided in mdx muscle fibers since they typically stained darkly. A total of 625 fibers per muscle were analyzed in each group (i.e. normal or mdx). Cross-sectional areas, whole fiber O D values and fiber core O D values of muscle fibers were compiled according to fiber type (type I, type Ila, type lib), muscle (EDL, soleus or diaphragm), mouse (1-5 for normal; 1-5 for mdx) and group (normal vs. mdx). The data were combined such that differences were examined between the corresponding muscles of normal and mdx mice. Cross-sectional area and O D differences among animals of each group and within a particular fiber type were also looked for. Interanimal differences within each group were analyzed using a fully crossed multiple analysis of variance ( A N O V A ) . Differences between fiber types of each muscle and between each group were accepted at a significance level of p<0.005 using a one-way A N O V A . Data in its expanded form are given in appendices 1-4. 35 R E S U L T S General Anatomy of the EDL, Soleus and Diaphragm The mouse E D L is a thin, pennate-shaped, anterior compartment leg muscle whose primary function is to extend the digits of the foot. It originates on the upper tibia and descends in the anterior leg between the tibialis anterior (medial) and the peroneus longus (lateral). At the level of the superior extensor retinaculum, the tendon first splits into two and then into four parts that descend under the inferior extensor retinaculum in the dorsum of the foot to eventually insert on the middle and distal phalanges of the toes. Upon dissection, the E D L appears white in color with its midbelly region lying in the upper one-third of the anterior compartment where it receives its nerve supply from a branch of the common peroneal nerve. The mouse soleus is a broad, fleshy, posterior compartment leg muscle lying immediately anterior to the gastrocnemius but arising entirely inferior to the knee. Functionally, the soleus acts in combination with the gastrocnemius to produce plantar flexion of the foot. It has a broad origin within the superior portion of the posterior compartment originating from the posterior head of the fibula and the tendinous arch of the intermuscular septum between the tibia and fibula. Its muscle fibers descend into a broad membranous tendon which fuses below with the deep surface of the tendon of the gastrocnemius muscle to form the calcaneal tendon. Distally, this strong tendon attaches to the middle part of the posterior surface of the calcaneus. In contrast to the E D L , the mid-belly portion of the soleus is located in the mid-to-lower leg where it receives its innervation from a muscular branch of the tibial nerve. Upon dissection, the soleus muscle appears deep pink to red in color. The diaphragm is a broad, thin, musculofascial sheet that moves air in and out of the lungs. It is the major muscle of inspiration, its contraction accounting for 36 most of the inspired volume of air by producing negative pleural pressure. Due to its complexity and broad attachments, the diaphragm has been divided into three major regions: the sternal region anteriorly which consists of two fleshy slips of muscle related to the xiphoid process, the costal region which consists of muscular slips and is associated with the inner surfaces of the costal cartilages and adjacent bones of the lower six ribs, and the crural region posteriorly which arises from the lumbar vertebrae. This portion of the diaphragm allows for the passage of the esophagus and aorta. Collectively, the three portions radiate inwards inserting into the central tendon. A l l regions are innervated by the phrenic nerve. It has been previously demonstrated that regional differences in muscle fiber types, sizes and orientations exist between these three portions of the diaphragm in the hamster (Reid et al., 1989). For this reason, and due to the scope of the study, only the costal region of the diaphragm was used for comparison between normal and mdx mice. Whereas at the macroscopic level the age-matched mdx mouse muscles were essentially identical in their anatomy, there was a slight increase in size when compared to their normal counterparts. Upon dissection, the soleus muscle of mdx mice was typically lighter in color (i.e. light pink) than the normal soleus. In addition, white lesions corresponding to sites of inflammation were often observed in the mdx muscles when viewed under the dissecting microscope. These lesions were most notable in the mdx diaphragm. Microscopic Appearance Low magnification cross-sectional views of normal and mdx E D L , soleus and diaphragm muscles stained for N A D H - T R are shown in Figure 1. A mosaic pattern of fiber staining was clearly revealed in the normal and mdx E D L . The soleus and diaphragm, on the other hand, exhibited less muscle fiber diversity in N A D H - T R -37 Figure 1 Low magnification views of NADH-TR-stained cryosections of the E D L , soleus and diaphragm from normal and mdx adult (32 wk) mice. x40. 38 39 stained sections in comparison to the E D L . Fibers within the soleus and diaphragm were typically moderate to dark in their staining properties. Fibrosis in the form of an increase in perimysial and endomysial tissue was moderate in the E D L and soleus, and more extensive in the diaphragm. Higher magnification views of serial sections from each of these muscles stained with H & E , mATPase and N A D H - T R are shown in Figures 4-6. In general, three types of fibers based on their N A D H - T R staining intensity could be discerned qualitatively in the E D L . Large, pale staining fibers corresponded to 'white fibers', small, dark staining fibers corresponded to 'red fibers', and fibers with intermediate staining properties corresponded to 'intermediate fibers'. A wider range of staining intensity and a clearer distinction of fibers was observed in the normal E D L (Fig. 4b) when compared to the mdx E D L (Fig. 4b'). In contrast to the E D L , the soleus of both normal and mdx mice exhibited a mixture of 'intermediate' and 'red' fibers (Figs. 5b, b'). This was also observed in the diaphragm (Figs. 6b, b'). The distinction between these fibers proved difficult, however, and was not as clear of a distinction as that in the E D L . Whereas muscle spindles were commonly observed in the E D L and soleus (Figs. 4, 5), these neuromuscular receptors were never encountered in the diaphragm (Fig. 6). Morphometric and Microphotometric Analysis Mean values and their standard deviations (±S.D.) for pooled fiber cross-sectional areas from the three muscles examined are presented in Table 1 and their frequency distributions are shown in Figure 2. As a whole, muscle fibers in the mdx E D L exhibited a statistically significant (p<0.001) 10% increase in cross-sectional area from a mean of 1680.4 u,m2 in normal to 1869.4 urn2 in mdx mice. Soleus muscle fibers of normal and mdx mice, on the other hand, did not show a significant 40 T A B L E 1 Mean cross-sectional areas (um2) and N A D H - T R optical density (OD) values (±SD) for total muscle fibers examined in the E D L , soleus and diaphragm of normal (N) and dystrophic (Mdx) mice. Data were compiled and analyzed according to animals (n=5) per group and according to total fibers (n=625). Core O D measurements excluded dense subsarcolemmal staining. NADH-TR Optical Density Area Whole Fiber Fiber Core n=5 mice n=625 fibers n=5 mice n=625 fibers n=5 mice n=625 fibers EDL N 1680.4 ±150.8 1680.4 ±728.0* 0.203 ±0.013* 0.203 ±0.132* 0.132 ±0.010 0.132 ±0.096 Mdx 1869.4 ±168.8 1869.4 ±1073.9 0.162 ±0.018 0.162 ±0.109 0.131 +0.019 0.131 ±0.096 Soleus N 2067.7 ±321.7 2067.7 ±590.6 0.218 ±0.015* 0.218 ±0.052* 0.149 ±0.012 0.149 ±0.040 Mdx 2081.9 ±301.3 2081.8 ±962.0 0.173 ±0.018 0.174 ±0.035 0.134 ±0.012 0.134 ±0.032 Diaphragm N 1055.6 ±106.9* 1055.6 ±460.8* 0.228 ±0.004t 0.228 ±0.056* 0.178 ±0.008* 0.178 ±0.057* Mdx 786.3 ±78.9 786.3 ±413.8 0.244 ±0.012 0.244 ±0.063 0.211 ±0.015 0.211 ±0.069 * p<0.005 between N and Mdx groups, as determined by a one way A N O V A . + p<0.05 between N and Mdx groups, as determined by a one way A N O V A . 41 Figure 2 Frequency distribution of muscle fiber cross-sectional area (in |im 2) for total fibers examined in normal and mdx E D L (a), soleus (b) and diaphragm (c). Mean cross-sectional areas for normal (white arrowheads) and mdx (grey arrowheads) are indicated. A total of 625 fibers was examined per muscle (n = 5 mice). 42 160 140 120 100 80 60 40 20 0 160 140 -120 -100 80 60 40 20 0 EDL v Soleus v © a 17771 Normal V 1 M d x ' b Diaphragm 1000 2000 3000 4000 Cross-sectional Area in um 2 5000 6000 43 difference in their mean cross-sectional areas. Whereas the frequency distribution of fiber areas in the normal and mdx E D L were relatively similar (Fig. la), the mdx soleus exhibited a wide range and broad distribution of fiber areas in comparison to the tightly-grouped pattern seen in the normal soleus (Fig. 2b). Some exceptionally large fibers (>4000 urn2) were present in both the mdx E D L and soleus (Figs. 2a, b). The most notable difference in fiber areas between normal and mdx muscles was found in the diaphragm. Fibers in the mdx diaphragm were, on the average, 26% smaller than those in the diaphragm of normal animals (786.3 u.m2 vs. 1055.6 u.m2). The frequency distribution histogram also revealed a corresponding shift in fiber numbers to a smaller fiber size (Fig. 2c). When compared to the E D L and soleus, muscle fibers in the diaphragm were 40-50% smaller in cross-sectional area in both normal and mdx conditions. Mean optical density values of NADH-TR-sta ined fibers from each of the muscles are presented in Table 1. Values were obtained from both whole fiber and fiber core tracings. With these two values it was possible to estimate differences in the oxidative capacity attributed to the subsarcolemmal population of mitochondria. Box plots representing the distribution of these data for total fibers from each muscle are shown in Figure 3. Whole fiber optical density measurements of N A D H - T R -stained fibers revealed a 20% reduction in total oxidative capacity in the mdx E D L and soleus fiber populations (Table 1, Figs. 2a, 2b). Fiber core measurements in the mdx E D L , however, did not show a similar decrease in value. In the soleus, a slight decrease from normal values was seen which showed significance at p<0.05. From these data, it was deduced that the decreased oxidative capacity in the mdx E D L and soleus muscle fibers was primarily associated with a reduction in the concentration and/or activity of the subsarcolemmal mitochondrial population. In contrast to the two hindlimb muscles, the mdx diaphragm showed an overall increase in oxidative capacity when compared to the normal diaphragm. 44 Figure 3 Box plots of the optical density of NADH-TR-stained muscle fibers in normal and mdx E D L (a), soleus (b) and diaphragm (c). Measurements were obtained from whole fiber and fiber core tracings. The thin midline of the box is the median, the thick line is the mean, the left border is the lower quartile and the right border is the upper quartile. Adjacent values are indicated by ends of whisker lines and + denotes minimum (left) and maximum (right). Significant differences (p<0.005) between normal and mdx mice are indicated with an asterisk (*). A total of 625 fibers were examined per muscle (n = 5 mice). 45 E D L Whole fiber * ^ / / y / / / / / / r / / / / / i / / / / / / / / 7 7 7 7 7 > * r—I I I I © a Fiber core ' ' ^ ^ • H I | — I 1 • 1 = 1 Mdx , I 1 - 1 1 1 1 1 1 1 1 1 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0:50 Soleus Whole fiber h-CZt * Fiber core I—V / /X /A—I * 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 Diaphragm Whole fiber h V///A///A —I II Fiber core -?ZZZZ&ZZZ2r I 1 I I 0.50 0.00 0.05 0.10 0.15 0.20 0.25 0.30 Optical Density (OD units) 0.35 0.40 0.45 46 Figure 4 Transverse serial cryosections of the normal (a-c) and mdx (a'-c') E D L stained with H & E (a, a'), N A D H - T R (b, b') and for demonstration of mATPase after acid (pH=4.5) preincubation (c, c'). Note the central nucleation and variation in extrafusal fiber size in the mdx E D L (a'). High N A D H - T R activity is present in small (red) muscle fibers while intermediate fibers have moderately high activity and large-sized (white) fibers have low activity (b, b'). The 'mosaic' oxidative staining pattern is more prevalent in the normal E D L (b). Type Ila (pale-staining) and type lib (intermediate-staining) extrafusal fibers predominate in this muscle (c, c'). Some darkly-stained intrafusal (bag) fibers of muscle spindles (arrowheads) are seen in c and c' for comparison. N o type I fibers are shown in these micrographs as they represented a very small proportion of fiber types (i.e. <0.5%) in the E D L . Type Ila (Ila), type Jib (lib). x350. Bar = 50 um. 47 48 Figure 5 Transverse serial cryosections of the normal (a-c) and mdx (a'-c') soleus stained with H & E (a, a'), N A D H - T R (b, b') and for demonstration of mATPase after acid (pH=4.5) preincubation (c, c'). A l l muscle fibers exhibit moderate to dark N A D H - T R staining (b, b'). Type I, type Ha and type LTb fibers are indicated in c'. Relatively few type lib fibers are present in the soleus. Equatorial regions of two muscle spindles are included in the fields of view. x350. Bar = 50 um. 49 50 Figure 6 Transverse serial cryosections from the costal region of normal (a-c) and mdx (a'-c') diaphragms stained with H & E (a, a'), N A D H - T R (b, b') and for demonstration of mATPase after acid (pH=4.5) preincubation (c, c'). A marked increase in connective tissue and reduction in muscle fiber size is observed in the mdx condition (a'). Muscle fibers stain moderate to high with N A D H - T R (b, b'). Type I, type Ila and type lib fibers are indicated (c, c'). x350. Bar = 50 um. 51 52 Mean optical density values of whole fiber and fiber core measurements are listed in Table 1 and a box plot of these data is shown in Figure 3c. Results from the fiber core measurements revealed a larger difference in optical density between normal and mdx diaphragm muscle fibers, suggesting that the main increase in oxidative capacity in these fibers was associated with the fiber core. Correlation of NADH-TR Staining with Type I, Ila and lib fibers To further examine fiber type populations within each muscle, serial sections were stained for mATPase activity after acid preincubation (pH 4.5). This allowed the comparison of the NADH-TR-stained sections with type I, type Ila and type lib fiber populations of each muscle. When stained with mATPase, a mixture of fiber types could be distinguished by their relative staining intensities in all muscles (Figs. 4c, c', 5c, c', 6c, c'). Preincubation at p H 4.5 revealed type I (dark-staining) and type II (pale-staining) fibers, the latter of which could be subdivided into two subtypes, type Ila (pale-staining) and type lib (intermediate-staining) fibers (Brooke and Kaiser, 1980; Doriguzzi et al., 1983). Within the E D L , mATPase staining after preincubation at p H 4.5 revealed two major fiber types, type Ila and type lib (Figs. 4c, c'). Darkly-stained type I fibers were rarely observed in both normal and mdx E D L muscles (<0.5%). Due to the their low numbers in both conditions, these fibers were excluded from subsequent calculations. A dark reaction was commonly observed in polar regions of intrafusal (bag) fibers of muscle spindles (Figs. 4c, c') and also served as a comparative measure of staining intensity for type Ila and lib fiber classifications. The proportions of each mATPase-based fiber type, their mean cross-sectional areas, and whole fiber and fiber core optical density measurements of N A D H - T R staining in the normal and mdx E D L are presented in Table 2. Frequency distributions of cross-sectional areas 53 T A B L E 2 Fiber type proportions (in %), mean cross-sectional areas (urn2) and N A D H - T R optical density values (+SD) for type Ila and type l ib fibers i n the E D L of normal (N) and dystrophic (Mdx) mice. Type I fibers were rarely encountered and were not included i n the tabulations. Data were compiled according to total number of fibers encountered i n the E D L of all 5 mice (number indicated i n brackets) or according to animals (n=5). Fiber core opt ical density measurements exc luded dense subsarcolemmal staining. NADH-TR Optical Densitv Fiber Tvpe lo Area Whole Fiber Fiber Core Type 1 N <0.5 Mdx <0.5 Type Ila N (n=359) 2164.3 ±511.3 0.105 ±0.048* 0.065 ±0.031 Mdx (n=317) 2315.6 ±1107.7 0.081 ±0.040 0.063 ±0.031 N (n=5) 57.4 +3.2 2162.1 ±203.1 0.105 ±0.013+ 0.065 ±0.011 Mdx (n=5) 50.7 ±8.3 2341.2 ±274.4 0.080 ±0.008 0.062 ±0.006 Type lib N (n=257) 1029.5 ±387.1* 0.333 ±0.085* 0.225 ±0.080 Mdx (n=301) 1410.1 ±814.7 0.245 ±0.094 0.201 ±0.089 N (n=5) 42.5 +3.3 1041.3 ±64.0* 0.332 ±0.008* 0.220 ±0.006 Mdx (n=5) 49.2 ±8.2 1421.3 ±155.9 0.242 ±0.025 0.198 ±0.027 * p<0.005 between N and Mdx groups, as determined by a one way A N O V A . + p<0.05 between N and Mdx groups, as determined by a one way A N O V A . 54 Figure 7 Frequency distributions of fiber cross-sectional area (in um2) for normal and mdx EDL, separated into type Ila and lib fiber types. Type I fibers were infrequently observed in the EDL and were, therefore, omitted from the tabulations. Mean cross-sectional areas for each fiber type in normal (white arrowheads) and mdx (grey arrowheads) muscles are indicated. 55 EDL © 100 80 Type Ila V a o <L> 60 40 20 J \///A Normal V i 1 Mdx • M ~ i — n -100 Type lib 1000 2000 3000 Cross-sectional Area in um 4000 2 5000 6000 56 for each fiber type in the E D L are given in Figure 7. In the normal E D L , type lib fibers were the smallest in size and comprised an average of 42.5% of the total fiber population. Type Ila fibers, in contrast, were the largest in size and constituted the majority (mean = 57.4%) of total fibers. The mean cross-sectional area for type lib fibers was 23% larger in the mdx E D L than in the normal E D L . No significant changes were noted in fiber type proportions in the mdx E D L , although a trend towards higher numbers of type Ila fibers was noticed. Box plots of O D values from whole fiber measurements for type Ila and Lib fibers in the E D L are shown in Figure 10a. Whole fiber optical density values showed a significant reduction in N A D H -TR staining (p<0.001) for both fiber types in the mdx E D L . A smaller reduction, however, was observed in the fiber core values (Fig. 11a) and was, therefore, attributed to differences in the subsarcolemmal mitochondrial population. Quantitative data from fiber types in the soleus separated into types I (dark-staining), Ila (pale-staining) and lib (intermediate-staining) are presented in Table 3 and frequency distributions of their cross-sectional areas are presented in Figure 8. Whereas the total numbers of type I fibers remained relatively unchanged between the normal (mean = 33.3%) and the mdx (mean = 37.0%) soleus, a more noteable shift in the type Ila and lib fiber populations were observed. In the normal soleus, type Ila and lib fibers represented, on average, 56.0% and 10.7% of the total fibers, respectively; whereas corresponding fiber types in the mdx soleus represented 45.1% (type Ila) and 17.9% (type lib). Significant differences in fiber size were noted in the type Ila and lib fibers but not in the type I fibers of the mdx soleus when compared to the normal soleus. Whole fiber optical density values of NADH-TR-staining were significantly lower in all fiber types of the mdx soleus (p<0.001); however, type Ila and type lib fibers exhibited the largest reduction in N A D H - T R staining intensity (Fig. 10b). A similar reduction in fiber core optical density measurements was also observed in these two fiber types (Fig. l ib) . 57 T A B L E 3 Fiber type proportions (in %), mean cross-sectional areas (um2) and N A D H - T R optical density values (±SD) for type I, type Ila and type lib fibers in the soleus of normal (N) and dystrophic (Mdx) mice. Data were compiled according to total number of fiber types encountered in the soleus of all 5 mice (number indicated in brackets) or according to animals (n=5). Fiber core optical density measurements excluded dense subsarcolemmal staining. NADH-TR Optical Density Fiber Tvpe % Area Whole Fiber Fiber Core Type 1 N (n=208) Mdx (n=231) 2163.4 2091.7 +563.0 ±923.7 0.168 0.155 ±0.029 ±0.031 0.115 0.120 ±0.025 ±0.030 N (n=5) Mdx (n=5) 33.3 +4.5 37.0 +6.8 2159.1 2040.2 ±322.1 ±416.6 0.167 0.155 ±0.008 ±0.016 0.115 0.120 ±0.007 ±0.012 Type Ila N (n=350) Mdx (n=282) 1929.6 2218.1 ±467.1* ±971.8 0.244 0.186 ±0.040* ±0.034 0.166 0.143 ±0.033* ±0.032 N (n=5) Mdx (n=5) 56.0 ±8.0 45.1 ±6.6 1951.1 2244.4 ±247.0 ±274.8 0.244 0.187 ±0.020* ±0.020 0.166 0.144 ±0.014+ ±0.015 Type lib N (n=67) Mdx (n=112) 2492.1 1718.6 ±915.2* ±928.9 0.240 0.180 ±0.052* ±0.035 0.164 0.141 ±0.048* ±0.032 N (n=5) Mdx (n=5) 10.7 ±8.0 17.9 ±3.0 2380.2 1728.4 ±574.8t ±238.4 0.246 0.180 ±0.022* ±0.016 0.166 0.141 ±0.020+ ±0.011 * p<0.005 between N and Mdx groups, as determined by a one way A N O V A . + p<0.05 between N and Mdx groups, as determined by a one way A N O V A . 58 Figure 8 Frequency distributions of fiber cross-sectional area (um2) for normal and mdx soleus, separated into type I, type Ila and type lib fiber types. Mean cross-sectional areas for normal (white arrowheads) and mdx (grey arrowheads) are indicated. 59 Soleus Type I V ® a v///\ Normal V i i Mdx • Type Ila Type lib V 1000 2000 3000 4000 Cross-sectional Area in um 2 5000 6000 60 T A B L E 4 Fiber type proportions (in %), mean cross-sectional areas (um2) and N A D H - T R optical density values (±SD) for type I, type Ila and type lib fibers in the diaphragm of normal (N) and dystrophic (Mdx) mice. Data were compiled according to total number of fiber types encountered in the diaphragm of all 5 mice (number indicated in brackets) or according to animals (n=5). Fiber core optical density measurements excluded dense subsarcolemmal staining. NADH-TR Optical Density Fiber Tvpe % Area Whole Fiber Fiber Core Type I N (n=56) 767.5 ±252.0 0.247 ±0.046 0.207 ±0.053 Mdx (n=115) 863.2 ±500.8 0.244 ±0.059 0.217 ±0.070 N (n=5) 9.0 +2.3 770.5 ±91.0 0.245 ±0.016 0.205 ±0.025 Mdx (n=5) 18.4 ±2.3 854.5 ±165.3 0.244 ±0.015 0.217 ±0.023 Type Ila N (n=348) 1257.6 ±493.3* 0.208 ±0.050* 0.154 ±0.048* Mdx (n=253) 752.5 ±368.9 0.244 ±0.061 0.208 ±0.067 N (n=5) 55.7 ±4.1 1256.2 ±162.3* 0.208 ±0.006* 0.154 ±0.006* Mdx (n=5) 40.5 ±10.4 766.6 ±79.6 0.243 ±0.018 0.207 ±0.019 Type lib N (n=221) 810.6 ±238.1 0.254 ±0.053 0.209 ±0.054 Mdx (n=257) 785.1 ±409.7 0.244 ±0.067 0.210 ±0.071 N (n=5) 35.4 ±2.9 808.4 ±54.6 0.255 ±0.014 0.209 ±0.019 Mdx (n=5) 41.1 ±11.2 779.9 ±82.9 0.247 ±0.015 0.213 ±0.018 * p<0.005 between N and Mdx groups, as determined by a one way A N O V A . 61 Figure 9 Frequency distributions of fiber cross-sectional area (um 2) for normal and mdx diaphragm, separated into type I, type Ila and type l ib fiber types. Mean cross-sectional areas for normal (white arrowheads) and mdx (grey arrowheads) are indicated. 62 100 80 Diaphragm Type I © i-i <D a 60 J o >-CD 1 40 20 V V//A Normal V i u Mdx • CO l-H CD o CD I 100 80 60 40 20 0 Type Ila ^ EiB^ / i I A 100 80 CO I M CD r2 60 MM o a I 40 20 V Type lib 1000 2000 3000 4000 Cross-sectional Area in um 2 5000 6000 63 Figure 10 Box plots of the optical density of NADH-TR-stained fibers in normal and mdx E D L , soleus and diaphragm separated into type I, type Ila and type lib fiber types. Optical density measurements were made on the whole fiber area including subsarcolemmal stain. See legend of Figure 3 for explanation of a box plot. Significant differences (p<0.005) between normal and mdx fiber types are indicated with an asterisk (*). 64 EDL Type I Whole Fiber ® a Y//A Normal T y p e I I a *Y*ZmZ^- 1 • # r — i Mdx *|—\ I | 1 1 • Type lib * I V//////XX///777777)t I 1 1 I 1 1 • 1 1 1 0.00 0.05 0.10 i i i i i 0.15 0.20 0.25 0.30 0.35 i 0.40 i 0.45 0.50 Soleus Type I - r T ~ " H • * b Type Ila • V-V/A\//A 1 . • * 1—1 1 1—1 * Type lib 1 V//AM//A 1 * • H l| 1 1 • * i i i 0.00 0.05 0.10 i i i i i 0.15 0.20 0.25 0.30 0.35 i 0.40 0.45 0.50 Diaphragm Type I • \y//yjy/A 1 • • i — i i l i 1 • c Type Ila . 1—V///X///A 1 * • 1 1 11 1 1 * Type l ib • 1 V//ty//\ 1 • • 1 1 ll 1 1 • • i i 0.00 0.05 0.10 1 1 1 1 1 0.15 0.20 0.25 0.30 0.35 Optical Density (OD units) i 0.40 1 0.45 i 0.50 65 Figure 11 Box plots of the optical density of NADH-TR-stained fibers in normal and mdx E D L , soleus and diaphragm separated into type I, type Ila and type lib fiber types. Measurements were made on fiber cores and excluded dense subsarcolemmal staining. See legend of Figure 3 for explanation of a box plot. Significant differences (p<0.005) between normal and mdx fiber types are indicated with an asterisk (*). 66 Fiber Core © EDL W Type I T y p e I I a ^ r ^ & - H • •unci—i • Type lib • I V////W//7777*-• I 1 I I — I a r777i Normal I 1 Mdx i 1 1 1 1 1 1 1 1 1 1 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 Soleus Type I Type Ila Type lib I—I II I — I i—i \ i—i * 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 Diaphragm Type I I—I I I Tvnella * 1 • • i i I • I 1 • * Type lib * I Y///A////A-• I—I H i-i 1 1 1 1 1 1 1 1 1 1 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 Optical Density (OD units) 67 Morphometric and microphotometric data of normal and mdx diaphragms are shown in Table 4 and in Figures 9, 10c and 11c. In contrast to the soleus, the diaphragm exhibited a relatively small number of type I fibers (mean = 9.0%). Their proportion increased by two-fold in the mdx diaphragm (mean = 18.4%). In general, type II fibers showed much more variability and range in their proportions between animals. In the normal diaphragm, type Ila fibers represented the majority of the fibers (mean = 55.7%) whereas type lib fibers constituted an average of 35.4% of the fiber population. The mdx diaphragm, in contrast contained relatively equal proportions of type Ila (mean = 40.5%) and type lib (mean = 41.1%) fibers. Mean cross-sectional areas of type I and type lib fibers in the diaphragm showed no significant differences between normal and mdx mice, however a significant decrease in size was noted in the type Ila fibers. Box plots of optical density values for type I, type Ila and type lib fibers in the normal and mdx diaphragms are shown in Figures 10c and 11c. The optical density values for each fiber type in the mdx diaphragm were virtually identical (Table 4). When compared to the normal diaphragm, the type Ila fibers of the mdx diaphragm were significantly higher in their N A D H - T R staining in both whole fiber (Fig. 10c) and fiber core (Fig. 11c) measurements. N o difference in N A D H - T R staining was found between type I and type lib fibers of the normal and mdx diaphragm. A n example of a typical necrotic lesion observed in the mdx soleus muscle is seen in Figure 12. Although these areas were observed in various regions of each mdx muscle, they were not included in the morphometric and microphotometric measurements since there was poor fiber discrimination and significant inflammatory cell accumulation. Necrotic muscle fibers were typically devoid of oxidative enzyme staining (Fig. 12b) but possessed some residual mATPase activity (Fig. 12c). At these sites, numerous neutrophils and macrophages surrounded the necrotic fibers with some of them appearing to occupy the sarcoplasm of these fibers. 68 Figure 12 Example of a myodegenerative region in the mdx soleus stained with H & E (a), N A D H - T R (b) and for demonstration of mATPase after acid (pH=4.5) preincubation (c). Several degenerating fibers (arrowheads) are surrounded by mononucleated cells, some of which appear to be present within the fibers. Note the lack of N A D H -TR staining in these fibers (b). x350. Bar = 50 um. 69 DISCUSSION The results of this study indicate that the oxidative capacity of skeletal muscle fibers in the E D L and soleus of adult mdx mice is lowered by approximately 20% in comparison to that in age-matched normal mice. This was not true in the diaphragm, however, where an overall higher oxidative capacity of the muscle fibers was observed in the mdx condition when compared to the normal diaphragm. These data support the hypothesis that the oxidative capacity of mdx muscle fibers in different muscles is affected to varying degrees depending on their location and function. The reduction in oxidative capacity of the two hindlimb muscles is in general agreement with the observations of Kuznetsov and coworkers (1998) on the mdx quadriceps muscle. These investigators, however, observed an even greater reduction (up to 50%) in mitochondrial function. Discrepencies in these values are possibly attributed to muscle-specific differences and/or methodologies used for determination of oxidative capacity. This group also reported that cardiac muscle in mdx mice did not exhibit alterations in mitochondrial function (Kuznetsov et al., 1988) suggesting that lower oxidative capacity was limited exclusively to limb musculature. The effect of this disease process on the oxidative capacity of muscle fibers in the diaphragm, however, has not been previously determined. Results from the present study indicated that muscle fibers in the mdx diaphragm, on average, contained a significantly higher oxidative capacity than that in the normal diaphragm. Microphotometric measurements of these mdx fibers revealed a 7% higher overall oxidative enzyme staining. From these findings, it was concluded that the oxidative capacity of non-injured muscle fibers in the mdx diaphragm was not compromised as a result of the disorder. Whole fiber and fiber core microphotometric measurements were made on NADH-TR-stained fibers to gain a better understanding of the regional distribution 71 of mitochondria within the muscle fibers, In general, the more oxidative type muscle fibers, i.e. the 'intermediate' and 'red' fibers, typically contained densely-packed regions of subsarcolemmal mitochondria in addition to mitochondria found between myofibrils in the central core. Based on comparisons between whole and fiber core measurements, changes in subsarcolemmal oxidative activity could be estimated. When analyzing these results, however, it should be noted that, even though differences in staining intensity were present, it was impossible to determine whether changes were present in the absolute volume density of mitochondria or whether the inherent enzyme activity of individual mitochondria was altered. In this study, it was assumed that the observed N A D H - T R staining was proportional to the volume content of mitochondria. In addition to an overall lower oxidative capacity in muscle fibers of the mdx E D L and soleus, changes within specific fiber types could be determined by correlating the NADH-TR-stained fibers with serially-sectioned, mATPase-stained fibers. After acid preincubation at p H 4.5, three fiber types were recognized based on the relative mATPase staining pattern that developed, and they were designated type I, type Ila and type lib fibers (Brooke and Kaiser, 1970; Doriguzzi et al., 1983). The E D L in both conditions was composed almost exclusively of pale (type Ila) and moderate (type Lib) staining fibers. Since a very small percentage of the E D L fibers in this study were classified as type I, they were not included in the comparison of fiber types. In general, type Ila fibers appeared to be low in oxidative capacity while type lib fibers were much higher and exhibited a wider range of activity between fibers. When compared to normal, the type lib fibers of the mdx E D L showed the largest reduction in oxidative capacity. This was primarily attributed to a decrease in subsarcolemmal staining since little difference was noted in their fiber core values when compared to these fiber types in normal E D L muscles. Lower oxidative capacity in the mdx soleus corresponded primarily to the type Ila and type lib fibers 72 with similar reductions in subsarcolemmal staining patterns. Type I fibers of the normal and mdx soleus muscles, in contrast, did not exhibit these differences. From these results, it is concluded that the major difference in oxidative capacities between normal and mdx hindlimb muscles is limited to the type II fiber population. In the mdx E D L where these fibers predominate, the type LTb fibers exhibited the greatest reduction in oxidative capacity. Subsarcolemmal and intermyofibrillar mitochondria have been considered by some researchers to be two separate types of organelles based on differences in their morphology and location (Gauthier, 1969; Muller, 1976). Moreover, it has been demonstrated that training of rat soleus muscles can specifically affect the subsarcolemmal population, of mitochondria by increasing this peripheral zone by as much as 53% (Muller, 1976). It is possible, therefore, that the lower oxidative capacity of muscle fibers in the mdx E D L and soleus may be a result of reduced physical activity of mdx mice due to the ensuing degenerative processes. A possible consequence of the lower oxidative capacity in the hindlimb muscles may cause a metabolic strain to be placed on the ATP-dependent processes within the muscle fibers. Basic physiological processes such as active ion transport and muscle contraction would, therefore, be compromised which may initiate and/or promote the degenerative process in muscle fibers. Although the mdx mouse serves as a genetic and biochemical animal model for human D M D , it has been reported that hindlimb muscle fiber necrosis in the mdx mouse is typically followed by successful regeneration with most muscles retaining their functional capacities (Dangain and Vrbova, 1984; Anderson et al., 1988). The diaphragm, on the other hand, has been shown to exhibit a significant age-related differential expression of dystrophy with respect to active tension generation (Dupont-Versteegden and McCarter, 1992) and a marked degenerative process that is characterized by reductions in elasticity and a seven-fold increase in 73 collagen density especially in the later stages of the disease (Stedman et al., 1991). Others have reported that the diaphragm of adult mdx mice undergoes a significant increase in the type I myosin heavy chain-positive fibers and a virtual elimination of the fast type Ilx/b fiber population (Petrof et al., 1993). These investigators have suggested that the mdx diaphragm responds to progressive muscle degeneration with a transition to a slower phenotype that is associated with both reduced power output and augmented muscle endurance. In the present study, a small increase in the numbers of type I fibers and an overall higher oxidative capacity in noninjured fibers was observed in the mdx diaphragm, which lends support to the findings of Petrof and coworkers (1993). It has become obvious that the unique structural and functional features of the diaphragm, coupled with its differential response to the degenerative process in the mdx mouse, clearly sets this muscle apart from the hindlimb musculature. The fact that muscle fibers in the mdx diaphragm were, on average, 26% smaller in cross-sectional area than their normal counterparts remains enigmatic. One possible explanation is that smaller fiber size may be a protective mechanism to reduce stress on the sarcolemmal membrane. Alternatively, the decrease in fiber size may reflect an elevated regenerative state of this muscle in dystrophy. It would be useful in future studies to examine the turnover rate of these fibers in the mdx diaphragm to test the latter prediction. In the normal mouse, the majority of muscle fibers in the diaphragm are of the type II variety (91%) implying that it is a relatively fast contracting muscle. In contrast to the diaphragm of larger mammals, muscle fibers of the mouse diaphragm are highly oxidative (Green et al., 1984). This feature has been attributed to their comparatively high breathing rate which ranges between 110 and 120 breaths/min (Crosfill and Widdicombe, 1961). Why an overall increase in oxidative capacity occurs specifically in the mdx diaphragm is unclear. One possible 74 explanation for these higher levels may be related to neural activity. It has been generally accepted that increased motor activity augments oxidative capacity in normal muscle (Maxwell et a l v 1973; Muller, 1974, 1976; Burke, 1981; Eisenberg and Salmons, 1981). A n obvious possibility to explain this increased oxidative capacity would be an increased breathing rate, although this has yet to be confirmed experimentally. Another possible reason for the higher oxidative capacity of muscle fibers may be that, since the mdx diaphragm undergoes a significant amount of degeneration/regeneration in its muscle fibers, and is also a continually active muscle, higher functional stress would be placed on viable fibers to maintain respiratory function. This increased load would , consequently, lead to a development of higher oxidative capacity within the fibers. It would be of interest to examine trained or functionally-overloaded muscles of the mdx mouse to test this prediction. Muscle wasting diseases that affect the oxidative capacity of muscle fibers have been noted in other mouse models of muscular dystrophy. Investigations on the d y / d y and d y 2 J / d y 2 J mouse models have revealed higher oxidative capacities in hind limb muscles including the tibialis anterior (Butler and Cosmos, 1977), gastrocnemius (Dribin and Simpson, 1977; Silverman and Atwood, 1982; Hargroder et al., 1986) and E D L (Ovalle et al., 1983; Parry and Parslow, 1981) muscles. It is not certain, however, if this holds true for the diaphragm in these mouse models. The fact that differences in the responsiveness of muscle fibers in the d y / d y and dy 2 J /dy 2 J mouse models to disease contrast to those observed in the mdx mouse hindlimb muscles suggests that energy metabolism is affected differently in these mouse models. The function of dystrophin, the protein product of the D M D gene (Hoffman et a l , 1987), is still not fully understood. Its absence in D M D and mdx skeletal muscle fibers, however, is currently believed to lead to a mechanical weakening of 75 the sarcolemma (Karpati et a l , 1986; Menke and Jockusch, 1991). This may be a result of a failure in linkage of the cytoskeleton to the sarcolemma via a glycoprotein complex and the extracellular matrix protein, laminin (Campbell and Kahl, 1989; Ervasti et a l , 1990; Ohlendieck et al., 1991; Ibraghimov-Beskrovnaya et al., 1992). Increased calcium-related differences either as a result of sarcolemmal disruption or abnormal activity of regulatory mechanisms have been proposed (Turner et al, 1988, 1991; Duncan, 1989), while some believe it may be a combination of the two mechanisms (Franco and Lansman, 1990). In addition, a mitochondrial deficiency in the calcium-binding protein, calmatine, has been implicated as causative factor for the degeneration of muscles in D M D and mouse models of dystrophy (Lucas-Heron et al., 1989; Lucas-Heron, 1995). It has been proposed that the deficiency of this mitochondrial calcium binding protein renders mitochondria more susceptible to degeneration and to subsequent fiber necrosis by affecting their ability to regulate calcium (Lucas-Heron et al., 1989; Lucas-Heron, 1995). From the body of evidence available, it has become obvious that no single specific mechanism has yet been attributed to the initiation of the disease process in D M D . Understanding the complex interactions and functional characteristics of cellular components in this degenerative process will provide new insights into prospective therapies for its treatment in humans. In conclusion, the results of this study indicate that the E D L and soleus muscles of adult mdx mice exhibit a significantly lower oxidative capacity in muscle-specific fiber-type populations. The majority of this difference is postulated to be due to changes in the subsarcolemmal population of mitochondria. 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Sladky and A . M . Kelly 1991 The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature, 352: 536-539. Turner, P.R. T. Westwood, C M . Regen and R.A. Steinhardt 1988 Increased protein degradation results from elevated free calcium levels found in muscle from mdx mice. Nature, 335: 735-738. Turner, P.R., P. Fong, W.F. Denetclaw, and R.A. Steinhardt 1991 Increased calcium influx in dystrophic muscle. J. Cell Biol., 115: 1701-1712. Wrogemann, K. and S.D.J. Pena 1976 Mitochondrial calcium overload: a general mechanism for cell-necrosis in muscle diseases. Lancet I, 672-673. 81 Chapter 2. Morphology and Quantitative Ultrastructure of Mitochondria and the Sarcotubular System i n Muscle Fibers of the Extensor Dig i to rum Longus, Soleus and Diaphragm i n Normal and M d x A d u l t Mice. I N T R O D U C T I O N In the past three decades, ultrastructural studies of skeletal muscle fibers have provided important insights into the diversif ied nature of fiber types w i t h i n mammalian muscles (Gauthier, 1967; Ogata and Murata, 1969; Schiaffino et al., 1970; Tomanek et al., 1973; Eisenberg and Kuda , 1976; Schmalbruch, 1979). Differences i n myofibrillar structure, Z line thickness, as we l l as content of mitochondria and the sarcotubular system have been used as criteria to distinguish three basic fiber types i n skeletal muscle fibers by electron microscopy (Eisenberg and Kuda , 1976). Other studies have shown that relative mitochondrial volume correlated w e l l w i t h histochemically-defined type I and type II muscle fibers (Shafiq et al., 1966; Ogata and Murata, 1969), and other reports implicated Z line thickness as the most reliable criterion (Garamvolgyi, 1972; Rowe, 1973). It has become evident that a combination of these ultrastructural parameters enables an accurate discrimination between fiber types of mammalian skeletal muscles. Depend ing on a g iven muscle and fiber type, variable numbers of mitochondria and membranous tubules and cisternae of the sarcotubular system are present i n the sarcoplasm. A t the ultrastructural level , the complexity of mi tochondr ia l structure coupled w i t h their in t racel lu lar d i s t r ibu t ion and orientation becomes evident. These organelles are aligned i n strategic sites wi th in the muscle fiber relative to both myofibrils and the sarcotubular system and provide metabolic energy for muscular contraction by producing A T P (Lieber, 1992). The sarcotubular system, on the other hand, surrounds myofibrils and comprises two 82 distinct membranous components termed the sarcoplasmic reticulum (SR) and transverse (T) tubules (Peachey, 1965). The SR of skeletal muscle fibers represents a special type of smooth endoplasmic reticulum comprising a continuous system of membrane-limited tubules and cisternae which form either a sheath or collar around each myofibril. T-tubules, on the other hand, are small-diameter surface invaginations of the sarcolemmal membrane that extend deep into the core of the muscle fiber. These two membranous networks come into close contact with one another at the so-called triad, a site that plays a critical role in excitation-contraction coupling in skeletal muscle fibers (Sandow, 1965; Caputo, 1978, Entman and Van Winkle, 1986). Numerous detailed descriptions of the mammalian SR network and its association with the T-tubule system and mitochondria using transmission electron microscopy (TEM) abound in the literature (Gauthier, 1967, 1969; Ogata and Murata, 1969; Schiaffino et al., 1970; Tomanek et a l , 1973; Eisenberg and Kuda, 1976; Schmalbruch, 1979). In comparison to other mammals, fine structural details of murine skeletal muscle fibers are relatively few in number (Shafiq et al., 1969; Luft and Atwood, 1971; Anderson et al., 1987). Even though Shafiq and coworkers (1969) revealed basic ultrastructural differences between muscle fibers in the fast-twitch extensor digitorum longus and the slow-twitch soleus muscles of mice little information regarding fine structure, spatial organization and content of the internal membrane systems was presented in their study. Moreover, since the discovery of the mdx mutant (C57BL/10ScSn) strain of dystrophic mice, an animal model for the human form of D M D (Bulfield et a l , 1984; Sicinski et a l , 1989), only a small number of studies pertaining to the ultrastructure of its skeletal muscle fibers have been undertaken (Bulfield et a l , 1984; Anderson et a l , 1987; Cullen and Jaros, 1988). In these studies, no information exists relating quantitation of intracellular membranes to functionally distinct fiber types. 83 The current hypothesis for myofiber necrosis in human D M D and in the mdx mouse predicts a primary sarcolemmal defect (Mokri and Engel, 1975; Carpenter and Karpati, 1979) that is believed to be related to the absence of the protein dystrophin (Hoffman et al., 1987). In contrast to human D M D , however, the degenerative process in the mdx mouse is mirrored by successful regeneration in its skeletal muscles (Anderson et al., 1988). How the mdx mouse is able to successfully regenerate, however, remains an enigma. In contrast to the sarcolemmal membrane lesions in human D M D muscle fibers, those in mdx fibers have been reported to be less deleterious and may not be the major factor leading to fiber necrosis (Cullen and Jaros, 1988). These investigators suggest that compensatory mechanisms in mdx muscle fibers may be responsible for their successful regeneration and these mechanisms remain to be elucidated. Since much of the research emphasis has been directed to the later stages of necrosis and degeneration of fibers in mdx skeletal muscle, it was of particular interest in the present study to examine the majority of muscle fibers that appear 'healthy' within mdx muscles. The overall lack of quantitative data and detailed descriptions on the organization of intracellular organelles in muscles of normal and mdx mice further prompted the current investigation. To answer the question of whether differences occur in the internal membranes of mdx muscle fibers when compared to those in normal muscles, morphometric measurements using a point counting system (Weibel, 1969; Eisenberg and Kuda , 1974) was employed on transmission electron micrographs to determine relative volume fractions of these intracellular membrane systems. This study will attempt to correlate data obtained from previous ultrastructural studies of skeletal muscle fibers of normal and mdx mice, as well as to provide new insights with morphometric data on mitochondrial and sarcotubular system content in different fiber types of normal and diseased skeletal muscle. 84 M A T E R I A L S A N D M E T H O D S TEM Processing A total of 10 adult male mice (25-32 wk) were used in this part of the study. Five were of the normal C57Bl/10ScSn strain and 5 were of the mdx mutant strain. Animals were bred and maintained under similar conditions and age-matched pairs were utilized for each experiment. The same three muscles examined in Chapter 1 (extensor digitorum longus (EDL), soleus and diaphragm) were used in this study to facilitate comparison with the previous results and with those of others (Bressler et al, 1983; Ovalle et al., 1983; Anderson et al., 1987; Cullen and Jaros, 1988; Stedman et al., 1991). Simultaneous processing of normal and dystrophic muscles was always performed in experiments to minimize potential differences in tissue preparatory procedures. Halothane anesthetized mice were perfusion-fixed via the left ventricle for 10 min with 100 ml of a 0.1 M sodium cacodylate buffer solution (pH 7.2) containing 1.5% glutaraldehyde and 1.5% paraformaldehyde. The E D L , soleus and costal region of the diaphragm were rapidly excised from normal and mdx mice and maintained at resting length during immersion in fresh fixative for 30 min on ice. The muscles were then cut into blocks 0.5 mm in length and reimmersed in fresh fixative for 2 hrs in a shaker on ice. Following several rinses in buffer, they were then placed in a buffered 2% osmium tetroxide solution (pH 7.2) for 2 hr at room temperature (RT), rinsed several times with distilled water and stained en bloc with 2% aqueous uranyl acetate for 1 hr (RT). Specimens were then washed several times with distilled water, dehydrated in graded ethanols (3 x 10 min each for 50, 70, 80, 90, 95, and 100% ethanols) and then placed in propylene oxide (3 x 15 min). The tissue blocks were then transferred to a 2:1 ratio mixture of propylene oxide:TAAB 812 resin (Marivac, Ltd., Halifax, NS) for 1 hr, a 1:2 ratio mixture for 1 hr, followed by pure T A A B 812 resin overnight. After infiltration, ten randomly selected blocks of each muscle were embedded in freshly prepared resin in flat rectangular molds. 85 Five blocks from each muscle were oriented transversely and five were oriented longitudinally. Molds were then placed in a 60°C oven for 8 hr to cure. Ultrathin sections (60-90 nm) were cut with a diamond knife on a Reichert Ultracut OmU4 ultramicrotome (Reichert-Jung, Vienna, Austria), picked up on acetone-cleaned copper grids, and contrast enhanced by staining briefly with uranyl acetate for 5 min followed by lead citrate for 5 min. Observations were made with a Philips E M 301 transmission electron microscope (Philips Electronics Inc., Eintoven, The Netherlands) operated at 60 kV and recorded on Eastman 35 m m fine-grain release positive film 5302. Morphometric Measurements In this study, the ultrastructural parameters chosen for measurement were fiber core mitochondria, SR, T-tubule and lipid volumes fractions, and Z line width. A l l measurements were obtained from fiber core regions of longitudinal sections. Peripheral regions of muscle fibers contained varying amounts of subsarcolemmal mitochondria depending on which areas of these regions were studied (i.e. near capillaries). Since these mitochondrial accumulations are highly variable depending on which regions are sampled in a given fiber, they were not included in the morphometric measurements in order to reduce sampling errors introduced by their presence. For each muscle, two of the longitudinally embedded blocks were selected at random and trimmed in a non-selective manner. Semi-thick sections (1 um) were stained with 1% toluidine blue at 60°C for 30 sec and examined for orientation by light microscopy. Only sections in which muscle fibers appeared in longitudinal section from one end of the section to the other were used. Uniform thin sections of a silver-gray interference color were then obtained by ultramicrotomy and contrast stained with uranyl acetate and lead citrate as described above. Muscle 86 fibers that either appeared to be degenerative or were less than 10 um in diameter were not included in the morphometric procedures. A total of 5 muscle fibers from each muscle (n = 5 mice) were chosen from the grid at random and two fields from each fiber were first photographed at low magnification for quantitation of mitochondria and lipid, and subsequently at high magnification within the center of the same regions for quantitation of SR and T-tubules. Z line measurements were made on the high magnification electron micrographs and represented in nanometers (nm). To ensure proper calibration of the electron micrographs, a JBS #401A replica grid with 21,600 lines/cm (J.B. E M Services Inc., Dorval, QE) was always photographed during sessions at the same magnifications to obtain consistent measurements since magnification settings on the electron microscope proved to be unreliable. For morphometric point counts, photographic prints measuring 18 x 20 cm were made for both low (final print magnification xl2,200) and high (final print magnification x28,725) power. The test grid consisted of a transparent sheet 21.6 cm x 28 cm with grid lines spaced 5 mm apart. The spacing of the test grid was dictated by the size and density of the profiles of interest in the micrographs. For both magnifications, the 5 mm spacings provided an accurate and reproducible point-count representation of the structures examined. The length of each square on the test grid was 0.414 um for low magnification electron micrographs (xl2,200), whereas for the high magnification electron micrographs (x28,725), the length of each square corresponded to 0.173 um. Area density was calculated using the formula A T = n x d 2 (coherent test system) where A T , n and d 2 represent sarcoplasmic (test) area, number of squares making up the test grid and length of square line, respectively (Weibel, 1969). The area density for each component of interest was determined by the number of times an intersection on the test grid landed on membranes or within enclosed membranes of each structure. According to the Delesse Principle, the area 87 determined from the point counts provides an estimate of the volume density for the component in a plane that transects the test volume. This method used for the measurement of volume density of intracellular organelles (Weibel, 1969) has been used extensively in studies of skeletal muscle (Eisenberg et al., 1974; Eisenberg and Kuda, 1975, 1976) for the quantitation of sarcoplasmic components. Since skeletal muscle fibers are highly organized and possess periodicity of structures due to their arrangement of myofibrils (i.e. anisotropic), the approach used in this study included the use of a fixed sectioning angle (i.e. longitudinal sections) and the test grid placed at an 'optimal angle' to yield the mean number of counts (Sitte, 1967). By placing the quadratic test grid at the 'optimal angle', this should give the correct mean value of the number of intersections for fully oriented surfaces. In counts of structures in longitudinal sections, the test grid was always placed at the optimal angle of 19° and 71° to the longitudinal axis of the muscle fiber. In practice, this was done by drawing a line at 19° and 71° relative to the lines on the test grid transparency and aligning it with the orientation of the axis of the contractile filaments on each micrograph. This method has been used by Eisenberg and coworkers (1976) for counts on electron micrographs of longitudinally-sectioned fibers and its validity has been previously confirmed through mathematical testing (Courant, 1937). In the event that a profile of the sarcoplasm may not always occupy the entire micrograph such as a portion of a nucleus, a subsarcolemmal zone, or the extracellular space, the test area of that fraction was calculated and subtracted from the sarcoplasmic area. The number of structures in the particular micrograph was then redefined with respect to the adjusted cytoplasmic area. A l l areas obtained from the morphometric procedures were then converted to a percentage of the total sarcoplasmic area tested. Data were compiled according to relative volume fractions of mitochondria and cut-off points were assigned for classification of muscle fiber 88 types into white (0-4.00%), intermediate (4.01-9.00%) and red (>9.01%) fibers. Averages and standard deviations (SD) for each parameter were calculated for the fiber types and statistical analysis was performed using a one way A N O V A for comparison between normal and mdx fibers. Differences were accepted as significant at p<0.05. Raw data from morphometric measurements are given in appendices 5-10. 89 R E S U L T S Observations in this study are focused mainly on the ultrastructural features of mitochondria and the sarcotubular system in three identifiable muscle fiber types found in the E D L , soleus and diaphragm of normal and mdx mice. Muscle fibers were classified as either 'white', 'intermediate' or 'red' fibers depending on the relative content of mitochondria. In addition, Z line thickness in longitudinal sections also served as a feature for distinction of the three fiber types. Normal ultrastructure of the E D L , soleus and diaphragm is described first followed by an account of these muscles in the mdx mouse. Morphometric data from each of the muscles in both conditions are considered together at the end of the Results to facilitate comparisons between normal and mdx muscle. Normal Morphology EDL A transverse section through a portion of the normal E D L showing the three basic fiber types is shown in Figure 13. Extrafusal fibers were closely packed into fascicles appearing as irregular, polygonal, cross-sectional profiles. White, intermediate and red fibers could be distinguished in the E D L based on muscle fiber diameter as well as on the density, distribution and organization of mitochondria. In transverse section, mitochondria in white fibers were limited exclusively to the lighter I band regions. These mitochondria exhibited highly branching, elongate shapes that coursed between myofibrils and partially encircled them on either side of the Z line (Fig. 14). In contrast, the intermediate and red fibers contained numerous circular profiles of more densely-stained mitochondria situated within A band regions (Fig. 13). Red fibers were distinguished by a relatively higher content of mitochondria and smaller cross-sectional diameters than those of the 90 intermediate fibers. Intermediate and red fibers also contained transversely-oriented mitochondria situated in the I band; these coursed in between myofibrils in a similar arrangement to those seen in the white fibers. Large circular profiles of mitochondria were seen within the I band and were typically connected to the thin, transversely-coursing mitochondria. These circular profiles, as will be discussed in more detail further on, corresponded to the columns of longitudinally oriented mitochondria. In addition to a higher content of mitochondria in the fiber core of both red and intermediate fibers, dense subsarcolemmal accumulations of mitochondria were arranged periodically along their perimeters. These were commonly observed near capillaries (Fig. 13) and were also adjacent to subsarcolemmal nuclei of the muscle fibers. These dense accumulations of subsarcolemmal mitochondria were never encountered in the white fibers. When viewed at high magnification, the mitochondria of white and red fibers were seen to vary morphologically, especially in the density of their internal cristae. Figure 14 is a comparative transverse sectioned view of a white and red fiber at their fiber peripheries. Mitochondria in the red fiber exhibit a more electron dense matrix and contain a higher density of cristae per mitochondrion than those in the white fiber. Mitochondria in intermediate fibers were intermediate in their morphology between red and white fibers with respect to the electron dense nature of their mitochondrial matrix and development of cristae. In muscle fiber cross-sections, the SR appeared as profiles of many small tubules measuring between 40 and 100 nm in diameter. Within the A band, these tubules were arranged in a single layer around myofibrils. In the I band region, on the other hand, the SR was more dense and exhibited multiple layers of small tubules in areas devoid of mitochondria (Fig. 14). At the junction of the A and I bands, triads were observed, consisting of two terminal cisternae and a centrally placed T-tubule. In contrast to the red fiber, white fibers exhibited an extensive 91 Figure 13 Low power electron micrograph of a transverse section of the normal E D L showing portions of a white (W), intermediate (I) and red (R) muscle fiber with an adjacent muscle spindle. Extrafusal fibers types were classified according to differences in mitochondrial density and arrangement. The large white fiber contains few mitochondria whereas the red and intermediate fibers contain numerous circular profiles of mitochondria in their fiber core as well as in subsarcolemmal regions. Capillary (c). x5000. Bar = lOum. 92 93 Figure 14 High power view of a white fiber (upper right) adjacent to a red fiber (lower left) in the normal E D L as seen in transverse section. Slender, serpentine, transversely-oriented mitochondria (Mi) are present in I band regions of the white fiber, while the large, spherical subsarcolemmal mitochondria (ssMi) are seen in the red fiber. A uniformly sized, delicate array of sarcotubules surrounds the myofibrils in the white fiber forming triadic couplings (circle) at the level of the A-I junction. Note the extensive subsarcolemmal SR network (ssSR) in the white fiber whereas relatively few sarcotubular elements are present in the subsarcolemmal region of the red fiber. Z line (Z). x36,000. Bar = 1 urn. 94 95 subsarcolemmal SR network (Fig. 14). This network comprised numerous circular profiles of tubules immediately underlying the sarcolemma. In longitudinal sections of all three fiber types, the SR network within the fiber core formed longitudinal sheets of anastomosing tubules that could be separated into three distinct regions based on their relationship to the sarcomeric banding of the myofibrils. These regions included the A band SR, the I band SR, and the terminal cisternae (or junctional SR) of the triads. A high magnification longitudinal view of a white fiber in the normal E D L (Fig. 15) at the level of the sarcomere shows these three regions of the SR. The A band SR formed an anastomosing network, the so-called 'fenestrated collar', in the mid-A band region of the sarcomere. This network can be revealed occasionally by T E M in longitudinal section when grazing sections are obtained along the length of a myofibril. Extending from the fenestrated collar of the A band SR is a less extensive network of longitudinally-oriented A band SR which terminates at the A-I junction. In contrast to the A band SR, the I band SR appeared as a looser array of interconnected tubules. These tubules were interposed with the transversely oriented mitochondria and were often intimately associated with their outer mitochondrial membranes. In longitudinal sections of white fibers, these mitochondria appeared as small circular profiles that ranged in diameter from 0.2 to 0.4 um. They were not always present in this location however, and in some I band intermyofibrillar spaces only SR elements were observed. At the A-I junction, a slender T-tubule measuring approximately 40 nm in diameter was usually flanked by two terminal cisternae emanating from the A and I band SR which, collectively, formed the triads. The ultrastructure of this junctional region is shown at higher magnification in Figure 22. In addition, small glycogen accumulations were often observed in the intermyofibrillar spaces. 96 Figure 15 H i g h magnification longitudinal section of a white fiber from the normal E D L showing the typical arrangement of mitochondria and the sarcotubular system relative to the sarcomere. A-band SR appears as a h ighly anastamosing, two-dimensional network of tubules, whereas the I band SR forms a loose network amongst the pairs of transversely-oriented mitochondria (arrowheads). Triads are composed of a central T-tubule l inked by periodic densities to the terminal cisternae of the A and I band SR. Note the pleomorphic mitochondrion (*) spanning the I band region. Z line (Z). x50, 000. Bar = 1 um. 97 98 Comparative views of sarcomeres from white, intermediate and red muscle fibers are shown i n Figure 16. When arranged i n register a notable difference is observed i n the thickness of Z lines between the three fiber types. White fibers exhibited thin Z lines, whereas red fibers of the normal E D L contained thicker Z lines. Intermediate fibers possessed Z lines intermediate i n thickness to those observed i n white and red fibers. The most notable difference between the three fiber types i n longitudinal sections was the content and orientation of mitochondria i n the intermyofibril lar spaces. Whereas white fibers contained small circular profiles of mitochondria at the I band level on either side of the Z line (Fig. 16a), intermediate (Fig. 16b) and red fibers (Fig. 16c) exhibited larger and more numerous, t ransversely-or iented mi tochondr i a as w e l l as l o n g i t u d i n a l co lumns of mitochondria that coursed parallel to the myofibrils. These columns consisted of variable numbers of mitochondria arranged end-to-end. Single mitochondria typically spanned at least one sarcomere; however, i n some cases they were observed to extend several sarcomeres i n length. Intermediate fibers contained thin mitochondrial columns that measured from 0.2-0.4 um i n diameter. Red fibers contained larger columns of mitochondria that were i n the range of 0.4-0.7 um in diameter. The transversely-oriented mitochondria of both intermediate and red fibers were similar i n size ranging i n diameter from 0.2 to 0.4 um w i t h some of them exhibiting continuity wi th the column mitochondria. Sarcotubular elements of intermediate and red fibers were similar i n disposition and general structure to those observed i n the white fibers. Due to the higher content of mitochondria i n the intermediate and red fibers, however, fewer SR tubules were present in those regions occupied by mitochondria. 99 Figure 16 Comparative longitudinal sections of a white (a), intermediate (b) and red (c) fiber in the normal E D L . White fibers are typified by thin Z lines and small transversely-oriented mitochondria whereas the intermediate and the red fiber have thicker Z lines and larger, more numerous mitochondria that course in both transverse and longitudinal (*) planes between the myofibrils. x28,000. Bar = lum. 100 101 Soleus Intermediate and red fibers predominated i n the soleus (Fig. 17) and both exhibited relatively similar fiber sizes. This slow-twitch muscle was characterized by an overal l abundance of mitochondria i n the majority of its muscle fibers. Mi tochondr i a of intermediate fibers were oriented i n both transverse and longitudinal planes and exhibited a dense array of cristae w i th in their matrices. Mi tochondr ia l columns of intermediate fibers ranged from 0.3 to 0.5 um i n diameter. Red fibers, i n contrast, contained much thicker mitochondrial columns ranging i n size from 0.5 to 0.9 um i n diameter. Small , granular electron densities were commonly observed i n the matrix of these mitochondria i n both fiber types. While sarcotubular elements i n both fibers showed the same basic arrangement as those i n the E D L , their density per sarcomere appeared to be lower than that i n the intermediate and red fibers of the E D L . Z lines of both fiber types i n the soleus were the thickest of al l muscle fibers examined i n this study and were often jagged i n appearance (Fig. 17). N o appreciable difference i n Z line thickness was noted between the two fiber types in the soleus; both averaged approximately 105 nm i n thickness. Dense accumulations of subsarcolemmal mitochondria were typically observed i n both fibers (Fig. 18) and were especially prominent i n the red fibers. A low magnification transverse section of an intermediate and a red fiber revealing these accumulations of subsarcolemmal mitochondria is shown i n the inset of Figure 18. Subsarcolemmal mitochondria were typically round-to-ovoid i n shape ranging i n size from 0.5 um to 2 um i n diameter. W h e n v iewed at higher magnification, some of these mitochondria were seen to exhibit elongated, snake-like portions that extended into the fiber core at the level of the I band (Fig. 18). 102 Figure 17 Comparative longitudinal sections of an intermediate (a) and red (b) fiber in the normal soleus. Mitochondria form thin, discrete columns in the intermediate fiber and thickened columns in the red fiber with occasional lipid droplets in between. Both fibers have thick, corrugated Z lines. x28,000. Bar = 1 um. 103 104 Figure 18 Transverse section of an intermediate fiber i n the normal soleus showing a collection of pleomorphic subsarcolemmal mitochondria w i t h densely-packed cristae. One mitochondrion has a thin, snake-like extension at the level of the I band into the fiber core (*). A low power view of the same intermediate fiber (I) and a neighbouring red fiber (R) is seen i n the inset. x28,000. Bar = 1 um. Inset x3300. Bar = 10 um. 105 106 Diaphragm For comparison, the salient ultrastructural features of muscle fibers i n the diaphragm are depicted in Figures 19-22. Similar to that observed i n the soleus, both intermediate and red fibers predominated i n the diaphragm while white fibers were absent. Figure 19a shows a low magnification view of the several red fibers beneath the mesothelial covering of the thoracic surface of the diaphragm. The sarcoplasm of these fibers was typically very rich i n mitochondria and contained an abundance of electron-lucent l i p i d droplets. Densely-packed subsarcolemmal mitochondria wi th compact cristae (Fig. 19b) were observed i n regions near capillaries. Both the intermediate and red fibers i n the diaphragm exhibited a complex, branching network of transversely-oriented mi tochondr ia at the level of the I band. Comparative transverse sections of the fiber cores from an intermediate and red fiber i n the d iaphragm are shown i n Figure 20. Red fibers differed from intermediate fibers not only by their higher content of mitochondria, but also by the abundance of l i p id droplets which appeared as clear spaces i n the sarcoplasm. It is noteworthy that these l i p i d droplets were always closely associated w i t h mitochondria (see Figs. 19-21). The red fibers of the diaphragm exhibited the thickest columns of mitochondria of all fibers examined; they were typically 1.0 to 1.5 um i n diameter. In these columns, mitochondria were stacked upon one another and appeared to fit together i n an interlocking, jigsaw-like pattern (Fig. 21b). Both fiber types i n the diaphragm exhibited moderately wide Z lines that ranged between 73.5 and 77.0 nm in thickness. The sarcotubular system i n muscle fibers of the diaphragm was similar i n structure and disposition to muscle fibers of both the E D L and soleus. In contrast to the soleus and E D L , however, the SR appeared to be more extensively developed i n the red fibers of the diaphragm (Fig. 21b), and to a lesser extent i n the intermediate fibers (Fig. 21a) i n this muscle. Well-defined arrays 107 Figure 19 Transverse section of the normal diaphragm at low (a) and high (b) magnifications. Figure 19a L o w magnification view of portions of three transversely-sectioned red fibers i n the normal diaphragm. The overlying mesothelium (Mes), a subjacent fibroblast and collagen are seen in the upper portion. x3700. Bar = 10 um. Figure 19b H i g h magnification of boxed port ion i n a showing the dense accumulations of subsarcolemmal mitochondria near a capillary i n two of these fibers. Note the compact, ordered array of cristae within these mitochondria. x20,000. Bar = 1 um. 108 109 Figure 20 Comparative transverse sections of an intermediate (a) and a red (b) fiber i n the normal diaphragm. Note the reticular pattern of mitochondria w i th in I band regions. Numerous l ip id droplets (L) are present i n the red fibers. xll,000. Bar = 1 u m . 110 I l l Figure 21 Comparative longitudinal sections of an intermediate (a) and a red (b) fiber i n the normal diaphragm. Transversely-oriented mitochondria of varying diameter are present i n the intermediate fiber. In b, a large co lumn of t ight ly-packed pleomorphic mitochondria (*) is seen i n between myofibrils of the red fiber. Note an elaborate sarcotubular system i n both fiber types and dichotomous branching of two t-tubules (circle) in the red fiber. L i p i d (L). x23,000. Bar = 1 um. 112 of A band SR were often encountered i n the fibers and extensive junctional regions (triads) were present. A t high magnification (Fig. 22), triads i n a red fiber from the normal diaphragm appeared to be structurally similar to those previously described in muscles of other species (Franzini-Armstrong, 1986), and i n longitudinal sections, evenly-spaced periodic densities between T-tubules and membranes of the terminal cisternae were clearly elucidated. These densities have been termed 'junctional feet' (Franzini-Armstrong, 1970; Somlyo, 1979) and span a gap measuring approximately 13 nm between the T-tubule and terminal cisternae. O n the inner surface of the junctional SR, filiform-like projections also showed a similar periodic arrangement relative to the junctional feet (Fig. 22b). Whi le the function of these structures remains to be elucidated, the calcium-binding protein, calsequestrin, has been shown to reside i n these regions of the terminal cisternae and may somehow be related to these projections (Franzini-Armstrong, 1986). 114 Figure 22 H i g h magnification views of triadic couplings seen in transverse (a) and longitudinal (b) planes in a red fiber of the normal diaphragm. Both x90,000. Bar = 0.5 um. Figure 22a A close association of the t-tubules (arrowheads) and terminal cisternae (TC) of the SR with a column-forming mitochondrion is seen in a. Figure 22b The junctional feet appear as periodically arranged densities between the t-tubule (arrowhead) and terminal cisternae (TC) of the A band and I band SR. Note an ordered array of filiform-like electron densities (arrows) on the inner surface of the junctional SR in the terminal cisternae. 115 116 Mdx Morphology EDL L o w magnification transverse (Fig. 23a) and longitudinal (Fig. 23b) views of the mdx E D L show that white, intermediate and red fibers corresponded i n structure to those seen i n the normal E D L . Commonly observed i n the dystrophic fibers, however, was the presence of centrally-located, euchromatic nuclei (Fig. 23a). M d x muscle fibers were more variable i n diameter and appeared more rounded than those of the normal E D L . They were separated by a prominent extracellular matrix consisting of collagen and inflammatory cells including macrophages, mast cells and tissue eosinophils. Longitudinal sections of 'healthy' appearing fibers showed that mitochondria are arranged in a similar fashion to those of the normal E D L (Fig. 23). Moreover, SR components in these fibers also exhibited normal structure. Alterations from normal morphology could also be detected i n many fibers of the muscle. One of the earliest ultrastructural changes was the distention of the SR, especially i n the I band SR elements (Fig. 24). Mi tochondr ia l and myofibril lar structure, however, appeared normal i n these fibers and the typical sarcomeric banding pattern was still preserved. For comparison, a longitudinal section of a middle stage degenerating fiber is shown i n Figure 25. Extremely distended SR vacuoles and distorted myofibrillar structure typified these fibers. Dur ing late stages of muscle fiber degeneration, the myofilaments became dissociated and lost their organized pattern (Fig. 26). In these fibers, the sarcolemma was poorly defined and the sarcoplasm contained aggregates of mi tochondr ia i n the core region. Mitochondria i n these fibers exhibited crystalline arrays between their cristae and also showed signs of disruption (Fig. 26b). Elements of the SR and T-tubule system, moreover, were unidentifiable in these fibers. 117 Figure 23 Transverse (a) and longitudinal sections (b) through portions of the mdx E D L showing white (W), intermediate (I) and red (R) fibers. Both x4000. Bar = 10 um. Figure 23 a Centrally located nuclei (*) are present i n the red and intermediate fibers. A degenerating fiber i n the lower left exhibits an amorphous sarcoplasm and lacks the normal myofibrillar structure seen in the neighbouring fibers. Several macrophages are also seen i n the upper left. Figure 23b A regular arrangement of mitochondria and myofibrils is present i n the three longitudinally-sectioned fibers. 118 119 Figure 24 Transverse section through a portion of a red fiber (upper right) and a white fiber (lower left) i n the mdx E D L . The white fiber exhibits signs of early degeneration by the presence of large, vacuolated SR components. SR elements i n the red fiber are small and uniform i n size. x34,000. Bar = 1 um. 120 121 Figure 25 Myofiber degeneration i n the mdx E D L . Disruption of normal myofibri l integrity and extreme dilation of SR elements (*) are observed i n the fiber on the left. The inset delineates a magnified area i n the fiber where a sarcotubular contact (circle) is observed. xl6,000. Bar = 1 um. Inset x53,000. Bar = 0.5 um. 122 123 Figure 26 Transverse section through a portion of a late stage degenerative fiber i n the mdx E D L seen at low (a) and high (b) magnification. The sarcolemma (arrowheads) is i l l -defined and both an overall loss of myofibri l lar structure and mitochondrial clumping occur i n the sarcoplasm. Mitochondria exhibit dense crystalline arrays wi th in their matrices and show signs of disruption (*). Capil lary (c), nerve (n). a, x5800. Bar = 10 um. b, x44,000. Bar = 1 um. 124 125 Soleus In the mdx soleus (Fig. 27), 'healthy' appearing fibers w i t h a regular arrangement of mitochondria and myofibrils were separable into intermediate and red fiber types and were comparable to those observed i n the normal soleus. Both fiber types i n the mdx soleus exhibited thick Z lines and a complement of mi tochondr ia and sarcotubular elements w i t h i n their fiber cores. Centra l nucleation was a common feature i n these fibers (Fig. 27b) and myosatellite cells were often observed at the periphery of muscle fibers sharing a common basal lamina (Fig. 28a). Numerous fibroblasts were also observed i n the extracellular spaces (Fig. 28a). In comparison to muscle fibers of the normal soleus, the subsarcolemmal region of muscle fibers i n the mdx soleus exhibited a more loosely-packed collection of spherical to ovoid mitochondria as we l l as varying numbers of lysosomes, Golg i complexes, and small tubular aggregates (Fig. 28b). In addition, fibers i n different stages of degeneration and regeneration were commonly observed; these were present in discrete regions of the muscle w i t h a similar occurrence to that observed i n mdx E D L . Abundant macrophages w i t h their characteristic lysosomal inclusion bodies were observed i n the interstitium and near late stage degenerative fibers (Fig. 29), some of wh ich occupied the amorphous sarcoplasm. The interstitium also contained tissue eosinophils and mast cells in regions adjacent to the necrotic fibers (Fig. 29). Diaphragm Muscle' fibers in the mdx diaphragm could be divided into intermediate and red fiber types (Fig. 30) and the ultrastructure of these 'healthy' appearing fibers was similar to that observed i n the normal diaphragm. Well-developed columns of mitochondria were a common feature of the fiber core and accumulations of subsarcolemmal mitochondria were also distributed around the fiber periphery. 126 Figure 27 Comparative longitudinal sections of an intermediate (a) and a red (b) fiber in the mdx soleus. A regular arrangement of mitochondria and myofibrils is seen i n both fiber types. Note the row of central nuclei i n the red fiber (*) which is a common feature of mdx skeletal muscle. Capillary (c). x6000. Bar = 5 um. 127 128 Figure 28 Transverse sections of the mdx soleus at low (a) and medium (b) magnification. Figure 28a A myosatellite cell is seen beneath the basal lamina (arrow) of an intermediate fiber and a neighbouring fibroblast (Fb) occupies the extracellular space. xl5,000. Bar = 1 u m . Figure 28b The subsarcolemmal region of a red fiber has a loosely packed array of spherical mitochondria, tubular elements, lysosomes (Ly) and a Golgi complex. x24,000. Bar = 1 um. 129 130 Figure 29 Micrograph of the mdx soleus showing several macrophages (*) surrounding and invading a degenerating muscle fiber (on the right). Two tissue eosinophils (E) are seen in close proximity to the macrophages. x!6,500. Bar = 1 am. 131 132 The sarcotubular system appeared to be unaffected in dystrophy in these fibers and was extensively distributed, especially in the red fibers where triadic and pentadic couplings of the SR and T-tubular system were often observed (Fig. 31). Small regenerating fibers were also commonly seen in the mdx diaphragm (Fig. 32). These fibers exhibited typical features of developing muscle including a loosely-packed array of myofilaments, primitive sarcotubular elements and central nuclei. Collections of perinuclear organelles most likely related to myofibril synthesis such as the rough endoplasmic reticulum also occupied the sarcoplasm of these regenerating fibers. Morphometric Analysis of Normal and Mdx Muscle Fibers Point counting procedures for the quantitation of both mitochondrial and lipid volume fractions in the fiber cores were performed on low magnification (xl2,200) prints of longitudinally sectioned muscle fibers. SR and T-tubule volume fractions were obtained from the same fibers by point counts on high magnification (x28,725) prints. A n example of a portion of the test grid used in this study and its orientation relative to the fiber axis at the optimal angle of 19° for low and high magnification prints is shown in Figure 33. For this study, the volume fraction of mitochondria was set arbitrarily at 0-4% for white fibers, 4.01-9% for intermediate fibers, and >9.01% for red fibers (Fig. 34). Their respective volume fractions of SR, T-tubules and lipid droplets together with Z lines thicknesses were then correlated with these three fiber types. Pooled numerical data from these three fiber types in normal and mdx E D L , soleus and diaphragm muscles are presented in Table 5. The muscle fibers sampled in the normal and mdx E D L exhibited a wide range in mitochondrial volume in their fiber cores. Frequency distributions of these values are shown in Figure 34a and the compiled data are presented numerically in Table 5. In the normal E D L , the volume fraction of mitochondria in the fiber core 133 Figure 30 Intermediate (I) and red (R) fibers of the mdx diaphragm seen in transverse (a) and longitudinal (b) section. Both x4000. Bar = 10 urn. Figure 30a A centrally-placed nucleus is seen in the red fiber (*) and numerous capillaries are situated in the surrounding endomysial connective tissue. Figure 30b A regular arrangement of myofibrils and mitochondria is seen in both fiber types. 134 135 Figure 31 Longi tudinal v iew of a red fiber from the mdx diaphragm. Large pleomorphic mitochondria and intermingling l ip id droplets form prominent columns between myofibrils. Note the extensive sarcotubular system including triads (arrowheads) and pentads in this fiber. A low magnification view of this fiber is shown i n the inset (in the upper left) for reference. x40,000. Bar = 1 um. Inset x2500. Bar = 10 um. 136 137 Figure 32 Transverse section of a regenerating fiber (indicated i n the inset w i th an asterisk) i n the mdx diaphragm. Early sarcotubular contacts such as triads (circles) are present in the sarcoplasm amongst a closely-packed array of myofilaments. Profiles of the rough endoplasmic ret iculum (arrow) are commonly observed, one of wh ich exhibits continuity wi th a terminal cisterna of a triad (arrowhead). A fibroblast (Fb) is seen i n the surrounding endomysial connective tissue. x37,000. Bar = 1 um. Inset xl600. Bar = 10 um. 138 139 Figure 33 White fiber in the normal E D L showing examples of the two magnifications used for morphometric measurements of longitudinally-sectioned muscle fibers. A portion of the test grid oriented at the optimal angle of 19° to the fiber axis is superimposed on the micrographs. Low magnification micrographs (a) were used to determine both mitochondrial and l ip id volume fractions, whereas high magnification micrographs (b) were used to measure SR and T-tubule volume fractions and Z line thicknesses. Images have been reduced slightly from their original sizes, a, xlO,000. b, x24,000. Bars = 1 um. 140 141 ranged from 2.47% to 12.22% whereas i n the mdx E D L a range of 2.27% to 9.44% was observed. The average content of mitochondria i n the white, intermediate and red fibers i n the normal and mdx E D L had similar values (see Table 5). A n inverse relationship of mitochondrial volume to SR volume was observed i n both normal and mdx E D L muscles. Respective values of the SR for white, intermediate and red fibers in the normal E D L were 9.22 ±1.00%, 8.33 ±0.63% and 7.64 ±0.51%, respectively, whereas in the mdx E D L these values were 9.30 ±1.29%, 8.37 ±1.28% and 7.88 ±0.88%. Between group comparisons revealed no statistically significant difference i n SR content between normal and mdx E D L fiber types. Between fiber comparisons in the normal E D L , however, revealed significantly higher SR i n white fibers when compared to intermediate and red fibers (p<0.05). Al though average SR values i n mdx E D L fiber types were similar to the values in fiber types of the normal E D L , these data d id not prove to be significantly different from one another. T-tubule volume fractions showed little difference between these two groups and no l ip id was encountered i n the muscle fibers sampled from either normal or mdx E D L muscles. Z line thickness of the three muscle fiber types i n the normal E D L showed dramatic differences wi th white fibers measuring, on average, 66.23 ±3.43 nm, intermediate fibers measuring 76.77 ±10.97 nm and red fibers measuring 98.76 ±7.69 nm (p<0.05). In the mdx E D L , a significant difference was noted i n Z line thickness when compared wi th these fibers i n the normal E D L . In the mdx E D L , white fiber Z lines measured 45.10 ±9.87 nm; Z line thickness of intermediate fibers i n the mdx E D L were not significantly different from that in the normal E D L wi th a wid th of 66.47 ±10.97. Al though it was noted that the Z line thickness i n red fibers of the mdx E D L was much thinner (67.67± 5.04) than those i n normal red fibers of the E D L , this was not considered va l id due to the low numbers of red fibers encountered i n the E D L in this study. 142 T A B L E 5 Compi led data from morphometric measurements of mitochondria (Mito), SR, T-tubules (T-T) and l i p i d content represented as a percentage of fiber core volume (±S.D.) i n white, intermediate and red fibers of the normal and mdx E D L , soleus and diaphragm muscles. Fibers containing 0-4.00% mitochondria were designated as 'white, ' 4.01-9.00% mitochondria were designated 'intermediate' and >9.01% mitochondria were designated 'red' fibers. Respective volume fractions of SR, T-tubules, l i p i d and Z line thickness measurements (in nanometers (nm)) were determined from these three fiber types and analyzed. Soleus and diaphragm muscles contained only intermediate and red fibers. Between group and between fiber differences were analyzed for significance using a one way A N O V A (p<0.05). EDL % M i t o % S R % T-T % L i p i d Z l ine (nm) Normal Whi te Intermediate R e d 2.89 ± 0 . 4 2 6.07 ± 0 . 9 7 10.71 ± 1 . 3 7 9.22 ±1 .00+ 8.33 ±0 .63+ 7.64 ±0 .51+ 0.56 ±0 .12 0.51 +0.11 0.46 ± 0 . 0 8 0 0 0 66.23 +3.43+* 76.77 +8.41+* 98.76 ±7.69+* Mdx W h i t e Intermediate R e d 2.87 ± 0 . 6 1 6.37 ± 1 . 3 7 9.41 ± 0 . 0 4 9.30 ±1 .29 8.37 ± 1 . 2 8 7.88 ±0 .88 0.61 ± 0 . 1 4 0.51 ± 0 . 1 4 0.58 ±0 .02 0 0 0 45.10 ±9.87+* 66.47 ±10.97+* 67.67 ±5 .04* SOLEUS Normal Intermediate R e d 7.71 ± 0 . 9 7 11.19 ±1 .96 5.51 ±0 .99 5.63 ± 0 . 9 4 0.45 ± 0 . 1 3 0.45 ± 0 . 1 2 0.21 ± 0 . 1 9 * 0.42 ± 0 . 2 2 102.82 ± 6 . 4 8 105.69 ± 3 . 2 0 Mdx Intermediate R e d 6.38 ± 1 . 2 1 10.07 ±0 .89 4.59 ± 0 . 6 5 4.87 ± 0 . 5 4 0.42 ±0 .10 0.36 ± 0 . 2 1 0.09 ± 0 . 0 6 * 0.15 ± 0 . 1 1 107.66 ± 4 . 0 4 106.85 ± 3 . 2 5 D I A P H R A G M -Normal Intermediate R e d 7.59 ±1 .09 12.06 +1.97 8.05 ±0 .89 7.98 ±0 .82 0.64 ±0 .12* 0.67 ±0 .20* 0.10 ± 0 . 0 9 0.20 ± 0 . 1 0 * 73.54 ±2 .79 77.00 ±4 .41 Mdx Intermediate R e d 6.39 ±1 .06 12.11 ±2 .04 7.01 ±0 .80 7.49 ±1 .49 0.42 ±0 .12* 0.48 ±0 .17* 0.07 ± 0 . 0 6 0.47 ± 0 . 2 3 * 70.60 ± 2 . 7 5 77.24 ±6 .30 * denotes s ignif icant difference be tween n o r m a l a n d m d x fiber types + denotes significant difference be tween fiber types w i t h i n musc le . 143 Figure 34 Frequency distributions of the volume fractions of mitochondria in the E D L (a), soleus (b) and diaphragm (c) in normal and mdx adult mice. Data were obtained from longitudinal sections of fiber cores by the point counting method of Weibel (1969) at a final print magnification of xl2,200 in 25 randomly selected fibers from five normal and five mdx mice. Dark vertical lines divide the frequency distributions of muscle fiber mitochondria volume fractions into white (0-4%), intermediate (4.01-9%) and red (>9.01%) fibers. 144 Volume Fraction of Mitochondria in Fiber Core (34) V//A Normal ] Mdx 4 5 6 7 8 9 10 11 12 13 14 15 16 17 15 % Volume Fraction of Mitochondria 145 Frequency distributions of normal and mdx mitochondrial fraction volumes in the soleus are shown i n Figure 34b. Muscle fibers i n the soleus were classified as either intermediate or red fibers. A notable shift i n the frequency distribution of mitochondrial volume fraction i n muscle fibers was observed i n the mdx soleus indicating that there was an overall reduction in the number of muscle fibers wi th a high content of mitochondria. In the normal soleus, intermediate fibers contained, on average, 7.71 ±0.97% mitochondria i n their fiber cores, whereas red fibers contained 11.19 ±1.96%. Average values for both fiber types i n the mdx soleus were slightly lower - intermediate and red fibers contained 6.38 ±1.21% and 10.07 ±0.89% mitochondria, respectively. SR volume fractions i n all soleus muscle fibers were significantly lower than those observed i n the E D L fiber types (p<0.05). Little difference i n SR content was noted between intermediate and red fibers of normal and mdx soleus muscles. SR volume fractions for intermediate and red fibers were 5.51 ±0.99% and 5.63 ±0.94% respectively, whereas i n the mdx soleus a slight reduction in their content was observed; their values were 4.59 ±0.59% and 4.87 ±0.54% i n intermediate and red fibers, respectively. T-tubule volume fractions were slightly lower in all fibers of the soleus when compared to the E D L . In both fiber types of the normal and mdx soleus, these values were i n the range of 0.36% to 0.45%. L i p i d droplets were encountered in both fiber types of the normal and mdx soleus. Intermediate fibers of the normal soleus contained 0.21 ±0.19% whereas red fibers contained 0.42 ±0.22%. Significantly less l i p i d was encountered i n intermediate fibers of the mdx soleus (0.09 ±0.06%) but was not found to be statistically different i n the red fibers (0.15 ±0.11%). Z line thickness of muscle fibers in the soleus was the widest of all fibers examined. Z lines measured 102.82 ±6.48 nm and 105.69 ±3.20 n m i n intermediate and red fibers of the normal soleus, 146 respectively. In the mdx soleus the Z line values were not significantly different at 107.66 ±4.04 nm and 106.85 ±3.25 nm, respectively. Frequency distributions of normal and mdx mitochondrial fraction volumes i n the d iaphragm are shown i n Figure 34c. Intermediate and red fibers predominated in the normal and mdx diaphragm. Intermediate fibers contained 7.59 ±1.09% and 6.39 ±1.06% mitochondria i n normal and mdx conditions, respectively. Red fibers, i n contrast, contained an extremely h igh content of mitochondria w i th average values of 12.06 ±1.97% and 12.11 ±2.04% i n normal and mdx diaphragms, respectively. SR content was not statistically different in fiber types of normal and mdx diaphragms. In the intermediate fibers, they constituted 8.05 ±0.89% and 7.01 ±0.80% of the fiber core volume i n the normal and mdx condition, respectively. In the red fibers, these values were 7.98 ±0.82% and 7.49 ±1.49%, respectively. T-tubule volume fractions were significantly different (p<0.05) between normal and mdx diaphragm fiber types. In the normal diaphragm, intermediate fibers contained a T-tubule volume fraction of 0.64 ±0.12%, whereas i n the mdx diaphragm, this value was significantly lower at 0.42 ±0.12% (p<0.05). A similar significantly lower T-tubule volume fraction was observed i n the red fibers of the mdx diaphragm (0.48 ±0.17%) when compared to red fibers of the normal diaphragm (0.67 ±0.20%). Like the two fiber types of the soleus, the intermediate and red fibers of the diaphragm also contained l ip id droplets i n their sarcoplasm. L i p i d values for intermediate and red fibers of the normal diaphragm were 0.10 ±0.09% and 0.20 ±0.10%, respectively, whereas in the mdx diaphragm these values were 0.07 ±0.06% and 0.47 ±0.23%, respectively. A significant difference was present between the normal red and mdx red fiber l i p id content (p<0.05). Whi le Z line thickness measurements were not statistically different i n either fiber types of the normal and mdx diaphragms, intermediate fibers possessed slightly thinner Z lines. They measured 73.54 ±2.79 nm in the normal and 70.60 ±2.75 nm i n the mdx. Red fibers 147 i n the diaphragm had slightly thicker Z lines which measured 77.00 ±4.41 nm and 77.24 ±6.30 i n the normal and mdx diaphragm, respectively. 148 DISCUSSION Since detailed ultrastructural studies of normal mouse muscle fibers are relatively few in number in the literature, it was anticipated that a better understanding of how these fibers compare to those in the more commonly studied animals such as rat, guinea pig, rabbit, and cat would be of beneficial value for future ultrastructural and experimental studies on skeletal muscles of mice. In this study, the E D L , soleus and diaphragm muscles were examined to clarify details of the spatial distribution and volume density of mitochondria and also the SR and T-tubule systems in muscle fibers. Moreover, this is the first report to reveal the quantitative morphology of these structures in adult mdx muscle fibers. Normal Ultrastructure The normal mouse E D L consisted of a heterogeneous population of muscle fibers that displayed a wide range of mitochondrial densities. Using volume fraction of mitochondria in the fiber core as a criterion, the ultrastructural distinction of three muscle fiber types was possible in this muscle. White fibers contained relatively few mitochondria and had thin Z lines whereas red fibers were rich in mitochondria and had thick Z lines. Intermediate fibers shared characteristics of both red and white fibers in that they contained moderate amounts of mitochondria and had Z lines that were intermediate in thickness to that observed in white and red fibers. Qualitatively, these morphological findings of the three basic fiber types are in agreement with those of the E D L in the rat (Schiaffino et a l , 1970). White fiber ultrastructure also corresponded well with those reported in previous studies on the mouse (Shafiq et al., 1969; Luff and Atwood, 1971) and other species (Schiaffino et al., 1970; Stonnington and Engel, 1973; Eisenberg and Kuda, 149 1975). In the white vastus muscle of the guinea p ig , w h i c h consists almost exclusively of the 'fast-glycolytic' type fiber, Eisenberg and K u d a (1975) reported a fiber core mitochondrial density of 1.47%, whereas, in the rat E D L , a value of 1.93% has been reported by others (Stonnington and Engel, 1973). These values are comparatively lower than the average mitochondrial volume of 2.89% observed in the white fibers i n the present study. Quantitative ultrastructural studies on the mitochondrial content i n mouse muscle fibers are, however, unavailable and no published studies are available for comparison. This relatively higher volume fraction of mitochondria i n the mouse white fibers may be indicative of species specific fiber type differences which may result from the variation i n metabolic rates between these animals. Al ternat ive ly , differences may have resulted from differences i n the sampling procedure, tissue preparation, identif ication and delineation of these membranous structures or data calculations. Morphometric studies on the internal membrane systems i n mouse skeletal muscle fibers are few in number, although one study has reported volume fraction values for the SR and the T-tubule system during postnatal development of the E D L and soleus muscles i n mice (Luff and Atwood , 1971). There were no data, however, on mitochondrial content or Z line differences wi th in specific fiber types of the indiv idual muscles. In a classical report on the comparative morphology of E D L and soleus muscle fibers in the rat, Schiaffino and coworkers (1970) revealed that both the fast-twitch E D L and slow-twitch soleus consisted of heterogeneous fiber populations that differed i n size, mitochondrial content, myoglobin concentration and thickness of Z lines. They also described the heterogeneity of muscle fibers of the fast-twitch E D L as being far greater than that observed i n the slow-twitch soleus. It has been postulated that the wide range i n fiber structure wi th in a muscle is a result of the composition of the motor units supplying that muscle (Burke, 1986). In muscles of adult animals, neural activity has been shown to play a primary role i n 150 the maintenance and plasticity of fiber type-specific profiles (Pette and Vrbova, 1985) which has been demonstrated by studies on the effects of cross-reinnervation of slow and fast muscles (Dubowitz, 1967; Sreter et a l , 1975; Reichmann et al., 1983). In contrast to the previous morphometric studies of mouse and rat E D L muscles (Shafiq et al., 1969; Luff and Atwood, 1971; Stonnington and Engel, 1973; Eisenberg and Kuda, 1975), few descriptions of the intermediate and red fiber types that are commonly observed in these muscles have been reported. Although regarded as a fast-twitch muscle which contains a high proportion of 'fast-glycolytic' (white) fibers, the mouse E D L was found in the present study to contain fibers rich in mitochondria that also possessed thick Z lines (~99nm). These were classified as red fibers and contained a volume density of mitochondria in the fiber core of 10.71% on average. Intermediate fibers, typified by an intermediately-sized Z line (~77nm), contained a moderate volume of mitochondria (6.07%) in their fiber cores. Thus, based on these results, three fibers in the E D L could be distinguished by their relative mitochondrial fraction volumes in their fiber cores and Z line widths. Significant differences were observed in the SR volume fraction of white, intermediate and red fibers of the E D L (p<0.05). Quantitative data from this study indicated that mitochondrial content is inversely related to the SR volume. White fibers showed the highest density of SR elements at 9.22%, whereas intermediate and red fibers contained significantly less SR at 8.33% and 7.64% respectively in the fiber core (p<0.05). Although there were no obvious morphological differences in SR ultrastructure and disposition relative to the myofibrils, quantitative differences were detected in overall SR content in these fibers by the point counting methods applied to longitudinal sections. These differences in SR content are likely attributed to the higher proportion of mitochondria occupying the intermyofibrillar spaces in red and intermediate fibers when compared to that in the white fibers. 151 Earlier fine structural reports on the soleus muscle of the mouse and of other larger species have commonly described this muscle as consisting of one general type of fiber, the slow-twitch oxidative fiber (Shafiq et al., 1969; Stonnington and Engel, 1973; Eisenberg et al., 1974). This, however, may be an oversimplification i n the mouse soleus since three fiber types based on mATPase staining can be distinguished (see Chapter 1). Microphotometric analysis of N A D H - T R stained fibers revealed that the acid-stable type I fibers were less oxidative than the type Ila and type l ib fibers, an opposite finding to that seen in type I, type Ila and type l ib fibers i n fast-twitch muscles. S imi lar observations on the his tochemical composition have been noted i n the soleus of the rat (Kugelberg, 1973). It is postulated that, i n the soleus of the mouse, the two metabolically-determined fiber types, intermediate and red, l ikely correspond to the type I and type II mATPase-defined fibers, respectively. In this regard, the soleus has been considered unique in that it exhibits some mATPase staining characteristics of fast-twitch muscles, although, from a physiological perspective, it exhibits slow-twitch properties. Due to these differences, it has been emphasized that the distinction between slow-twitch muscle fibers of the soleus and the slow fibers present in a fast-twitch muscle be recognized when interpreting physiological data (Gauthier, 1986). A noteable ultrastructural difference i n muscle fibers of the soleus was the low SR volume density i n both the intermediate and red muscle fibers when compared to the intermediate and red fibers of the E D L and diaphragm. Soleus muscle fibers contained an average of 5.51% and 5.63% SR i n the fiber cores of intermediate and red fibers, respectively. In contrast, the respective amount of SR in these fibers of the E D L were, on average, 8.33% and 7.64%. This difference i n SR content is consistent w i t h physiological data on the contraction speeds and relaxation times of soleus and E D L muscles (Anderson et al., 1988) and has been implicated as one of the major ultrastructural differences that sets slow-twitch and 152 fast-twitch muscles apart from one another (Schiaffino et al., 1970; Luff and Atwood , 1971). In addition to differences i n SR volume density, evidence of functional and structural diversity i n the calcium transport mechanisms of isolated SR membranes from fast-twitch and slow-twitch muscle fibers have provided further clues to understanding the physiological properties of this organelle i n these two basic types of twitch fibers (Sreter and Gergely, 1964; He i lmann et al . , 1977). Ca lc ium accumulating capacities of isolated SR from fast-twitch fibers have been reported to be 4 to 11 times greater than i n SR of slow-twitch fibers (Hei lmann et al., 1977). Similar differences i n the concentration of the calcium ATPase pump i n the SR have been demonstrated in situ through immunoelectron microscopic studies of these respective muscle fibers (Dulhunty et al., 1987). Thus, it has become more evident that a combination of structural and molecular differences exist i n the SR of fast- and slow-twitch muscle fibers of mammals. Fine structural studies on the muscle fibers of the diaphragm have been generally performed on the rat, rabbit or cat (Gauthier, 1969; Gottschall , 1980; Rambourg and Segretain, 1980; Ki larsk i and Sjostrom, 1990). In the rat diaphragm, ultrastructural classification of fiber types has been approached by a cluster analysis method by combining various parameters wi th in a fiber (Gottschall, 1980). In Gottschall 's (1980) study, two subpopulations were defined us ing a stepwise discriminant analysis, the most important parameters for discr iminat ion being mitochondrial volume and Z line wid th whereas other parameters were of minor importance (eg. SR, T-tubules). In one population, the fiber core volume density of mitochondria averaged 5.97% and contained thinner Z lines, whereas i n the other populat ion there was an average of 13.2% mitochondria i n the fiber core and moderately thick Z lines. Similar to Gottschall's (1980) findings, quantitative data from the present study revealed a b imodal dis t r ibut ion of fibers i n the d iaphragm based on 153 mitochondrial volume density, although, i n the mouse, there appeared to be no clear dist inction of fiber type using Z line thickness as a criterion; Z lines of intermediate fibers averaged 73.54 nm and those of red fibers averaged 77.00 nm. Moreover, i n contrast to the soleus, the SR volume fraction was substantially higher in the diaphragm muscle fibers suggesting that these fibers are better suited for faster contraction speeds and relaxation times. Of interest, however, was the significant difference i n l i p i d which was always higher i n the mitochondrial-rich red fibers. Whether this classification of two fiber types based on these parameters relates to physiological properties must be determined since mitochondria and l i p i d relate primari ly to the metabolic capacity of the fiber and do not include differences i n contractile activity associated w i t h the myofilaments. Further studies using immunoelectron techniques w i l l shed new light on molecular differences i n contractile function and how they relate to membrane systems of the muscle fiber. Mdx Ultrastructure Fine structural changes i n mdx muscles documented i n previous studies have described the presence of degenerative and regenerative foci consisting of single fibers or groups of fibers (Bulfield et al., 1984; Anderson et al., 1987; Cul len and Jaros, 1988). In the present study, these foci were consistently observed i n the adult muscles of mdx mice and were characterized by fibers i n various stages of degeneration and regeneration. The earliest detectable sign of degeneration appeared to be dilat ion of the SR. Focal dilation of the SR can appear i n many disorders of muscle fibers including periodic paralysis and human D M D (Engel and Banker, 1986). This ultrastructural change is also common i n denervated fibers (Engel and Stonnington, 1974), although it appears to be the first indicative change i n fibers of the mdx mouse. In middle stages of degeneration, fibers exhibited stretched and disoriented myofibrillar structure w i t h accentuated dilat ion of SR. 154 Late stages were characterized by myofibril disassembly resulting i n an amorphous appearing sarcoplasm w i t h no indication of striations. Invasion of macrophages into the sarcoplasm and the presence of other mononucleate cells were often observed either wi th in and nearby these necrotic fibers. The findings from this study support those of others on the degenerative process i n the mdx mouse (Bulfield et al., 1984; Anderson et al., 1987; Cullen and Jaros, 1988). The morphometric measurements on mdx muscle fibers i n this study placed emphasis on non-necrotic fibers in the mdx muscles, i.e. those without any gross degenerative changes. In these muscle fibers, a strikingly similar overall structure and volume fraction of their internal membrane systems was noted. Centrally nucleated fibers were almost always encountered, although this is a common feature of a number of myopathic and experimental conditions such as denervation or transplantation of normal muscles; their presence is thought to be indicative of regenerative activity of the muscle fiber (Engel and Banker, 1986). Observations i n this study indicate that mdx fibers undergo normal transformations to their respective fiber types i n the three muscles fol lowing regeneration. This suggests that neural activity is not impaired in the dystrophic condition. In experimental studies that have examined the effects of nerve stimulation, muscle fibers fail to differentiate histochemically and also exhibit alterations i n their ultrastructural specializations (Askanas et al., 1972). In other types of murine and human dystrophies, mitochondrial alterations have been described (Silverman and Atwood, 1982; Ovalle et al., 1983; Nishino et al., 1998). In mitochondrial myopathies, intramitochondrial crystalloids (Kamieniecka and Schmalbruch, 1980) were observed wi th in large subsarcolemmal clusters of mitochondria and i n some mitochondria of the fiber core. In addit ion, dense, spherical intramitochondrial granules measuring 10-30 nm i n diameter, a common feature i n normal mitochondria, can become exceptionally large i n mitochondrial 155 myopathies. Moreover, matrix granules in giant mitochondria can attain sizes up to 80 n m i n diameter and are often edge-shaped like crystals (Kamieniecka and Schmalbruch, 1980). In the present study, abnormal mitochondria were only observed i n grossly degenerative fibers that d isp layed alterations i n their myofibrillar structure, SR elements or a combination of the two. In these fibers, the matrix granules of mitochondria were large and irregular and adjacent cristae were occasionally fused by an electron dense layer. These alterations, however, are most likely secondary to the primary insult to the muscle since they were not observed in the other 'healthy' appearing fibers. It is predicted that the higher occurrence of matrix granules i n mitochondria of degenerating fibers is related to an excessive sequestering of cations present in the sarcoplasm by mitochondria. In conclusion, the results from T E M observations i n the present study indicate that skeletal muscle fibers of the normal E D L are separable into white, intermediate and red fiber types based on mitochondrial content i n the fiber core and Z-line thickness. O n the other hand, fiber types i n the normal soleus and diaphragm consisted of intermediate and red types based on mitochondrial and l ip id contents; no significant difference was noted i n their Z line widths i n either fiber type from each muscle. In the mdx condition, the earliest ultrastructural alteration i n muscle fibers was dilation of the SR followed by later degenerative processes including myofibril lar disorientation and large mitochondrial granule formation. 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Milhorat 1969 A n electron microscope study of fibre types i n normal and dystrophic muscles of the mouse. J. Anat., 104: 281-293. Sicinski, P., Y. Geng, A.S. Ryder-Cook, E .A. Barnard, M . G . Darlison and P.J. Barnard 1989 The molecular basis of muscular dystrophy i n the mdx mouse: a point mutation. Science, 244: 1578-1580. Silverman, H . and H . L . A twood 1982 Increase of muscle mitochondrial content wi th age i n murine muscular dystrophy. Muscle Nerve, 5: 640-644. Sitte, H . 1967 Mophometrische Untersuchungen an Zel len. In: Quantitative Methods i n Morphology. E.R. Weibel and H . Elias (eds.). Springer-Verlag, N e w York Inc., N e w York, pp 167-198. Somlyo, A . 1979 Bridging structures spanning the junctional gap at the triad of skeletal muscle. J. Cel l B i o l , 80: 743-750. Sreter, F .A . and J. Gergely 1964 Comparative studies of the Mg-activated ATPase activity and Ca uptake of fractions of white and red muscle homogenates. Biochem. Biophys. Res. Comm., 16: 438-43. Sreter, F . A . , A . R . Luff and J. Gergely 1975 Effect of cross-reinnervation on physiological parameters and on properties of m y o s i n and sarcoplasmic reticulum of fast and slow muscles of the rabbit. J. Gen. Physiol. , 66: 811-821. Stedman, H . H . , H . L . Sweeney, J.B. Shrager, H . C . Haguire, R . A . Panettieri, B. Petrof, M . Narusawa, J .M. Leferovich, J.T. Sladky and A . M . Kel ly 1991 The mdx mouse d iaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature, 352: 536-539. Stonnington, H . H . and A . G . Engel 1973 N o r m a l and denervated muscle. A morphometric study of fine structure. Neurology, 23: 714-724. Tomanek, R.J., C R . Asmundson, R.R. Cooper and R.J. Barnard 1973 Fine structure of fast-twitch and slow-twitch guinea pig muscle fibers. J. Morphol . , 139: 47-66. Weibel, E.R. 1969 Stereological principles for morphometry i n electron microscopic cytology. Int. Rev. C y t o l , 26: 235-302. 160 Chapter 3. T h r e e - D i m e n s i o n a l Ul t ras t ruc tu re of M i t o c h o n d r i a and the Sarcotubular System i n Skeletal Muscle Fibers of N o r m a l and M d x A d u l t Mice as Revealed by High-Reso lu t ion Scanning Electron Microscopy. I N T R O D U C T I O N Three-dimensional modeling of intracellular membrane systems i n skeletal muscle fibers has been previously achieved through a number of methodologies including systematic analysis of ultrathin serial sections by T E M (Bakeeva et al., 1978, 1981; Kayar et al., 1988), high-voltage electron microscopy of thick sections (Kirkwood et al., 1986, 1987), selective enhancement by specialized staining methods (Rambourg and Segretain, 1980; Scales and Yasumara, 1982; Peachey and Franzini-Armstrong, 1983; Hayashi et al., 1987), or by freeze-fracture replica techniques (Bray and Rayns, 1976; Franzini-Armstrong, 1986). The disadvantages of these methods, however, are the tedious reconstruction of th in sections, the relatively low resolution images obtained from thick sections and l imited depth of field offered by T E M . High-resolution scanning electron microscopy (HRSEM) is a major technical contribution to the field of microscopy that can span the gap between these T E M methodologies. It allows an immediate grasp of the spatial organization of intracellular structure by providing a sense of depth and three dimensionality. U n t i l recently, conventional scanning electron microscopy (SEM) was used mostly for surface observation of cells wi th relatively little attention to intracellular structures, due i n most part to physical limitations i n microscopic instrumentation (Hollenberg and Erickson, 1973). Over the past two decades, however, the resolving power of SEMs has steadily improved w i t h innovations i n microscope design. Modern H R S E M s equipped wi th field emission sources and objective lenses of very 161 short focal length are now commercially available and have a resolving power approaching that of conventional T E M (Hollenberg et al., 1989; Tanaka et al., 1989). The high resolution of this k ind of instrument not only permits the analysis of fine structure on surfaces but also allows investigation of intracellular structures when addit ional techniques for examination become available. Var ious preparative procedures have been used for specific or selective observations of internal structure, and depending on the information desired, the investigator can subsequently choose the appropriate procedure (Tanaka, 1989; Lea et al., 1992). In early S E M studies of skeletal muscle (Ishikawa et al., 1983), it was practically impossible to expose, just by fracturing methods alone, cellular structures and organelles usually embedded i n the fixed sarcoplasm of the muscle fiber. Both the myofiber interior and its surface can be better visualized when tissues are treated sequentially and often repeatedly wi th tannic acid and osmium tetroxide (Os0 4 ) prior to critical-point drying (Tanaka and Mitsushima, 1984). The tannic acid-Os0 4 treatment, also known as the conductive staining method, is especially useful for H R S E M because metal coating can be much reduced. In addition, freeze fracturing of previously fixed tissue blocks followed by prolonged treatment wi th dilute O s 0 4 provides excellent preservation of intracellular membranous organelles i n fractured surfaces of cells. First introduced by Tanaka and Naguro (1981), the dilute O s 0 4 extraction technique, or the Os0 4 -dimethylsulphoxide ( D M S O ) - O s 0 4 method (O-D-O method), allows for membranous structures inside of fibers to be disclosed i n relief by selectively extracting excess cytoplasmic matrices from the fractured surfaces of cells. A further modification of this technique included an initial fixation step wi th dilute aldehydes (0.5% glutaraldehyde and 0.5% paraformaldehyde) to better preserve ultrastructural morphology of the tissue prior to extraction (Tanaka and Mitsushima, 1984). Subsequently, the procedure has become commonly known as the aldehyde prefix O-D-O method, or the A - O - D - O method. 162 Further modificat ions of this procedure have evo lved since its first application to demonstrate the three-dimensional morphology of mitochondria and the SR i n rat and human extrafusal muscle fibers (Ogata and Yamasaki, 1985a, 1985b, 1997), intrafusal fibers of rat muscle spindles (Ogata and Yamasaki, 1991, 1992), and rat myocardial fibers (Ogata and Yamasaki, 1990), as wel l as i n avian (Ogata and Yamasaki, 1993) and amphibian (Ogata and Yamasaki, 1987, 1989) muscle fibers. To date, however, there have been no studies on the three-dimensional organization of mitochondria and the sarcotubular system of skeletal muscle fibers i n the mouse. Furthermore, comparative three-dimensional ultrastructural studies of these organelles w i t h H R S E M i n functionally-diverse muscles or i n myodegenerative states of skeletal muscle are few i n number (Goto et al., 1990; Kawahara et al., 1991). The three main objectives of this study are: 1) to confirm earlier observations by H R S E M of mitochondria and sarcotubular systems of the three mammalian skeletal muscle fiber types (white, intermediate and red), 2) to extend our knowledge of the spatial distribution of these structures i n three functionally-diverse muscles (the fast-twitch extensor digitorum longus (EDL), the slow-twitch soleus and the diaphragm) of the mouse, and 3) to compare the H R S E M structure of these internal membrane systems i n muscle fibers of normal and mdx mice. Muscle fibers in the E D L , soleus and d iaphragm exhibit d ivers i ty i n their morpholog ica l and histochemical features (see Chapter 1) as wel l as i n their ultrastructural architecture and volume proportions of mitochondrial and sarcotubular elements (see Chapter 2). Accordingly, H R S E M was used to extend our previous results obtained by light microscopy, histochemistry and T E M to three-dimensional H R S E M . In order to visualize the spatial arrangement of both the SR and mitochondria i n muscle fibers, the A - O - D - O procedure (Tanaka and Mi t sush ima , 1984) was used to extract nonmembranous components of the sarcoplasm. The present study illustrates 163 morphological variability of these organelles in different muscle fiber types of normal and mdx mouse muscles. 164 M A T E R I A L S A N D M E T H O D S Specimens were prepared according to the method originally devised by Tanaka and Mitsushima (1984) for selective cytosolic extraction of nonmembrane-bounded components. A total of five normal adult mice (25-32 wk) of the C57BL/10ScSn strain and five age-matched mdx mutant mice of the same strain were used in this study. E D L , soleus and diaphragm muscles were rapidly excised following ventricular perfusion with 0.5% paraformaldehyde and 0.5% glutaraldehyde in 0.067 M cacodylate buffer, p H 7.4, for 5 min. The muscles were then cut into small pieces (1-2 m m 3 blocks) and fixed for an additional 45 min in fresh fixative at 4°C. Following aldehyde fixation, the tissue blocks were rinsed with cacodylate buffer (3 x 15 min) and placed into 1% O s 0 4 in cacodylate buffer for 2 hr at 4°C. After rinsing with buffer (3 x 15 min), the specimens were successively immersed in 25% and 50% D M S O for 30 min each at room temperature (RT), quick-frozen in isopentane cooled to -196°C in liquid nitrogen. The specimens were then transferred to a freeze fracturing block immersed in liquid nitrogen. The freeze fracturing block consisted of a solid brass cylinder with a specially-machined, 1 cm deep well on one end which served to contain the frozen tissue blocks and fractured pieces during the cleavage process. The frozen muscle blocks were fractured by gently tapping with a precooled, single-edged razor blade while continuously immersed in l iquid nitrogen. Fractured pieces were then thawed in 50% D M S O at 20°C and rinsed in buffer until the D M S O was completely removed. After rinsing, specimens were fixed in a 1% O s 0 4 buffered solution for 1 hr at RT followed by a brief rinse in buffer. They were then placed into 0.1% O s 0 4 in buffer for 3-7 days at RT to remove nonmembranous components of the sarcoplasm. Midway through the extraction process, muscle samples were rinsed with buffer (3 x 15 min) and a new 0.1% O s 0 4 solution was 165 added to the specimen containers. Because of the delicate nature of both the sarcotubular elements and mitochondria, care was always taken when handling specimens during the processing procedure (i.e. minimal agitation). Extraction of the fractured surfaces could be indirectly monitored by the formation of black precipitate in the dilute O s 0 4 solution. Following extraction, the specimens were rinsed well with buffer to remove all traces of precipitate and then fixed for 1 hr in a buffered 1% O s 0 4 solution at 20°C. They were washed with distilled water, conductive stained with 2% tannic acid for 2 hr, washed with buffer (3 x 15 min), and additionally fixed for 1 hr at 20°C in a buffered 1% O s 0 4 solution. After rinsing well in distilled water, they were dehydrated in a graded series of ethanols and then critical-point dried in a Balzers Union C P D 020 (Manchester, N H ) using liquid carbon dioxide as the intermediary fluid (Lea and Ramjohn, 1980). The fractured surface of specimens was identified under a dissecting microscope and specimens were then carefully mounted in various orientations on precleaned aluminum stubs with conductive silver paint. Specimens were then sputter coated with a thin layer of gold (3-5 nm) using a Nanotech SEMPREP 2 (Manchester, UK) and argon gas as the ionizing plasma. Observations were made on a field-emission type Hitachi S-4200 H R S E M (Hitachi, Tokyo) and recorded on Type 55 Polaroid positive/negative film (Cambridge, M A ) . Stereo pairs were obtained by tilting specimens ±8°. 166 R E S U L T S H R S E M observations i n this part ot the study pertain to the three-dimensional ultrastructure and spatial dis t r ibut ion of mi tochondr ia and the sarcotubular system i n three distinguishable fiber types i n the normal and mdx E D L , soleus and diaphragm. Skeletal muscle fibers were classified as either 'white,' ' intermediate' or 'red' by their relative content of mi tochondr ia (Fig. 35). Differences i n fiber types were also based on muscle fiber diameters, the spatial distribution of mitochondria i n fiber cores and the presence of subsarcolemmal accumulations of mitochondria. In H R S E M prepara t ions , the th ree -d imens iona l v i s u a l i z a t i o n of mitochondria and the sarcotubular system i n skeletal muscle fibers was greatly enhanced by selectively extracting myof ibr i l s and other nonmembranous components from the sarcoplasm. To obtain sufficient depth of extraction, it was necessary to leave specimens i n dilute O s 0 4 for 5 to 7 days, since specimens examined at shorter extraction times (2-4 days) contained fine granular material associated w i t h membranous elements resul t ing i n poor d i sc r imina t ion of intracellular structure. A t 7 days extraction, the optimal time used for extraction process i n this study, the depth of extraction below the fractured surface of the fibers was approximately 10-20 um (as determined by T E M observations of resin-embedded specimens). Moreover, membranous components i n these fractured surfaces were relatively free from granular material. Thus, the effect of dilute O s 0 4 over prolonged periods of time promoted dissolution of filamentous material while at the same time also maintained and preserved intracellular membranes. A n exception to the general statement that all non-membranous components were extracted from the sarcoplasm was the fact that the nuclei of muscle fibers remained intact. They typically appeared wel l preserved and contained their content of both 167 heterochromatin and euchromatin, as well as nucleoli. Further elaboration on properties of osmium that allow it to act as both a fixative and an extraction agent for certain elements of the cell will be dealt with in more detail in the Discussion. Three-Dimensional Ultrastructure of Normal Muscle EDL . In transverse fractures of the normal E D L , a well-defined pattern of polygonal spaces (previously occupied by myofibrils) was observed in white and intermediate fibers, whereas a looser, more irregular pattern was seen in red fibers (Fig. 35). These polygonal patterns corresponded to differences in size, shape and arrangement of myofibrils which typically had cross-sectional dimensions in the range of 1 to 2 um. The structure of this 'honeycomb' in extracted fibers consisted of both the sarcotubular system and mitochondria; both of these organelles contributed to formation of a tube like, membranous 'sheath' that enveloped myofibrils along their length. Mitochondria were recognized by their larger size in comparison to the sarcotubules in addition to the presence of internal cristae in fractured-open profiles. In white fibers, the sarcotubular network predominated in the walls of the myofibril 'sheaths' and often obscured the thin, tranversely-oriented mitochondria which were located in the I band (Fig. 36a). Intermediate fibers also exhibited an extensive and well-defined sarcotubular honeycomb that contained circular profiles of fractured mitochondria amongst the sarcotubular network (Fig. 36b). These circular profiles represented the column-forming mitochondria and were larger and more numerous in the red fibers than in the intermediate fibers. They were rarely observed in the white fibers. Intermediate and red fibers could also be distinguished by accumulations of subsarcolemmal mitochondria which were distributed periodically around the periphery of the fibers (Fig. 35). 168 Figure 35 L o w magnification H R S E M view of a transversely fractured portion of the E D L i n the normal mouse showing profiles of a white (W), intermediate (I) and red (R) fiber. Nonmembranous sarcoplasmic components were extracted by exposure to dilute O s 0 4 (0.1%) for 7 days at room temperature. A well-defined, honeycomb-like, membranous network is observed i n the white and intermediate fibers, whereas a looser, more irregular network is seen i n the red fiber. Circular profiles of column-forming mitochondria are larger and more numerous i n the red fiber than in the intermediate fiber, whereas none are observed i n the white fiber. Note the accumulations of mi tochondr ia (*) i n subsarcolemmal regions of both the intermediate and the red fiber. x7200. Bar = 5um. 169 170 Figure 36 High magnification transverse views comparing a white (a) and an intermediate (b) fiber in the normal E D L . The highly developed sarcotubular system of both fibers envelopes the polygonal-shaped myofibrils which are extracted and appear empty. Two thin, column-forming mitochondria (*) and a transversely-oriented, I band-limited mitochondrion (arrows) have been fractured open to reveal their internal cristae. x45,000. Bar = 1 um. A low magnification view of these two fibers is included in the inset for reference. Inset x860. Bar = 25 um. 171 In longitudinal fractures of white fibers i n the normal E D L , the subdivisions of the SR network into A band, I band and junctional SR were readily observed and, i n addition, the spatial orientations of mitochondria could be better appreciated (Fig. 37a). Whi te fibers i n the normal E D L contained thin, transversely-oriented mitochondria that coursed between myofibrils to partially encircle them at the level of the I band. Observations by T E M have previously revealed that these mitochondria are paired circular profiles that straddle the Z line (see Chapter 2). By H R S E M , it was noted that these mitochondria were embraced by the I band SR i n a clasp-like fashion (Fig. 37). O n the other hand, very few longitudinally-oriented mitochondria were encountered i n the white fibers. SR profiles i n both the A band and I band regions i n white fibers were relatively uniform i n size and measured between 40 to 60 n m i n diameter. The A band SR consisted of an extensive sheet-like network of anastomosing tubules that formed a well-defined fenestrated collar. The fenestrated collar consisted of a tight, coalescing network of SR tubules that was restricted to the m i d - A band level of the sarcomere. Longitudinally-oriented components of the SR emanating from either aspect of the fenestrated collar extended to the triadic couplings where they joined the terminal cisternae at the triad. In white fibers of the E D L , the terminal cisternae formed an almost continuous cistern adjacent to T-tubules. Between myofibrils at the level of the A - I junction, slender T-tubules averaging 40 nm i n diameter were interposed by two adjacent terminal cisternae of the SR which collectively formed the triads. Bare, or nonjunctional, regions of the T-tubules were rarely observed i n these fibers; they were typically associated wi th two terminal cisternae of the SR along their length. In I band regions, the SR tubules embraced the mitochondria i n a clasp-like fashion and, at the level of the Z band, typically formed a continuous cistern (Fig. 37). In I band regions not occupied by mitochondria, the SR formed a compact three-dimensional array of anastomosing tubules that extended from triad 173 to triad. Dilatations or vesiculations of the SR i n fibers of the E D L were seldom seen except for the moderately distended regions at the terminal cisternae of triadic couplings. The most striking difference noted between the three fiber types i n the E D L was the content and orientation of mitochondria i n the intermyofibrillar spaces. In general, all fibers contained transversely-oriented mitochondria at the level of the I band. These I band mitochondria were small in diameter i n white fibers, slightly larger i n intermediate fibers, and largest in diameter i n red fibers. In addition, intermediate and red fibers possessed longitudinal columns of mitochondria along the lengths of the myofibrils (Fig. 37b). In these columns, single mitochondria were observed to span several sarcomere lengths and exhibited distinct side-branches at the I-band level. When cleaved open, these mitochondria were seen to possess a tightly-packed network of cristae, the majority of which were tubular i n shape. A s a rule, subsarcolemmal regions of white fibers were devo id of mitochondrial accumulations. O n the other hand, intermediate fibers contained a few subsarcolemmal accumulations whereas red fibers contained numerous aggregations of these subsarcolemmal organelles (Fig. 38). Subsarcolemmal regions devoid of mitochondria contained an anastomosing SR network that consisted both of a discrete, fenestrated zone corresponding to the level of the A band and a discontinuous array of enlarged tubules at the level of the I band (Fig. 38a). Small vesicles were also dispersed on the inner sarcolemmal surface between the SR tubules. T-tubules were extremely difficult to identify i n this region since observation of the inner surface required a shear fracture immediately adjacent to the subsarcolemmal surface, which most l ikely severed these minute tubules from their origin at the sarcolemma. The dense accumulations of mitochondria observed at the periphery of red fibers consisted of a tightly packed arrangement of spherical-174 Figure 37 Comparative longitudinal views of a white (a) and red (b) fiber in the normal E D L . Mitochondria are typically oriented as transverse pairs (arrowheads) within the I band of the white fiber. Red fibers contain thickened columns of mitochondria (*) that course longitudinally and exhibit continuity with the mitochondria at the level of the I band. In both fiber types, the SR of the A band (A) is composed of anastomosing sheets of tubules that form a fenestrated collar at mid-A band levels. Longitudinally-oriented tubules of the A band SR extend towards the A-I junction and coalesce to form the terminal cisternae. In the I band (I), a three-dimensional array of SR tubules courses between and around the mitochondria in a clasp-like fashion. x37,500. Bar = lum. 175 Figure 38 Subsarcolemmal regions of an intermediate fiber (a) and a red fiber (b) i n the normal EDL. Figure 38a O n the internal sarcolemmal surface, the SR forms discrete fenestrations (*) at the level of the A band (A) and a discontinuous array of enlarged tubules at the level of the I band (I). Small vesicles are also dispersed on the inner surface of the sarcolemma. Mitochondrion (Mi). x42,000. Bar = l u m . Figure 38b This view shows a subsarcolemmal accumulation of large, tightly-packed, spherical mitochondria i n a red fiber. Mitochondria have been fractured open to reveal their internal cristae which are densely packed and exhibit both a tubular and cisternal morphology. x70,000. Bar = l u m . 177 178 to-ovoid mitochondria containing an elaborate array of internal cristae that exhibited both a tubular and a cisternal morphology (Fig. 38b). Soleus By H R S E M , muscle fibers i n the normal soleus could be classified as either intermediate or red based mostly on their content of mitochondria (Fig. 39). Intermediate fibers were characterized both by pairs of relatively large-diameter mitochondria at the I band level and thin mitochondrial columns coursing parallel to myofibrils and spanning a distance of 1-2 sarcomeres (Fig. 39a). The SR i n the two fiber types of the soleus d i d not show the same degree of organization as that observed i n the muscle fibers of the E D L . In contrast to the tight, coalescing tubules seen i n fenestrated collars of fibers in the E D L , the SR in the fibers of the soleus at mid -A band regions was characterized by wide and dispersed fenestrae. In addition, the I band SR was discrete and was seen to closely intermingle wi th the transversely oriented mitochondria. Red fibers of the soleus contained an extensive mitochondria l network composed of thickened, elongate columns wi th dimeric side-branching at the level of the I band (Fig. 39b). The SR i n these fibers was less abundant than that i n the intermediate fibers and also displayed a large variability i n both shape and size, measuring 35-160 nm i n diameter. Intermyofibrillar sheets of A band SR were poorly defined and contained large, distended portions wi th in the A-band and at the sites of triadic couplings. W i t h i n the I-band, thin extensions of SR from the terminal cisternae of the t r iad coursed over and i n between the pairs of mitochondria and formed a single cistern at the level of the Z-band. Three-dimensional v iewing of the T-tubules commonly revealed bare, or non-junctional, regions wh ich were observed coursing through the intermyofibrillar spaces and around the columns of mitochondria at the A- I junction (Fig. 39b). 179 Figure 39 Comparative longitudinal views of an intemediate fiber (a) and red (b) fiber in the normal soleus showing the typical arrangement of sarcotubular elements and mitochondria. Both x27,000. Bar = 1 um. Figure 39 a Large, transversely-oriented, I band-limited mitochondria predominate with some longitudinal columns spanning A band regions (*). The SR of the A band exhibits wide fenestrae whereas I band SR is intimately associated with mitochondria. Several T-tubules are indicated with arrows. A band (A), I band (I). Figure 39b This view shows elongate, pleomorphic columns (*) of mitochondria with dimeric side branches (arrowheads) at I bands. A discrete vesicular pattern of the SR is seen and T-tubules are indicated by arrows. A band (A), I band (I). 180 Subsarcolemmal mitochondria were also a common feature of both the intermediate and the red fibers of the soleus. A n example of a these spherical-to-ovoid subsarcolemmal mitochondria is seen in Figure 40. Although most of these mitochondria conformed to a spherical or ovoid shape, a small proportion also exhibited thin, snake-like extensions into the intermyofibrillar regions of the fiber at the level of the I band (Fig. 40b). Diaphragm The costal portion of the diaphragm is composed of relatively small diameter muscle fibers that, by H R S E M could be classified as either intermediate or red based on mitochondrial content. At low magnification, a survey view of a transversely fractured diaphragm reveals an upper (pleural) surface and lower (abdominal) surface (Fig. 41). Both surfaces are covered by a mesothelium that exhibits small, apical microvilli on its outer surface. In transverse fractures, intermediate and red muscle fibers in the diaphragm (Fig. 41) exhibited distinct honeycomb patterns of their membranous components. Circular profiles corresponding to column-forming mitochondria were small in intermediate fibers and large in red fibers. Morever, both fibers in the diaphragm displayed distinct aggregations of subsarcolemmal mitochondria. When viewed in the longitudinal plane, intermediate fibers in the diaphragm contained an abundant and transversely-oriented array of mitochondria at the I band with thin mitochondrial columns (Fig. 42a). The SR was well-developed and showed a prominent fenestrated collar at mid-A band levels. Junctional SR was extensive and bare regions of the T tubules were rarely observed. Red fibers similarly exhibited a well-developed SR, the only obvious difference between these fibers being their thick and prominent mitochondrial columns (Fig. 42b). Closely 182 Figure 40 L o w magnification view (a) of portions of two red fibers (R) i n the normal soleus that have been fractured transversely. Dense accumulations of subsarcolemmal mitochondria are indicated (*). Note also the large mitochondrial profiles i n the fiber core and the discrete nature of the sarcotubular network. x8800. Bar = 5um. A n enlargement of the boxed region (b) shows mitochondria (below) wh ich are typically spherical to ovoid i n shape. These mitochondria have densely-packed cristae and occasionally exhibit snake-like extensions (arrowhead) into the myofibrillar region of the fiber. x28,000. Bar = l u m . 183 Figure 41 L o w magnification transverse v iew of the complete thickness of the normal diaphragm (inset above) and a higher magnification view of several muscle fibers that have been transversely fractured (below). Intermediate (I) and red (R) fibers predominate i n the diaphragm, the majority of which exhibit dense accumulations of subsarcolemmal mitochondria (*). The mesothelial surface covering the diaphragm is seen above. Blood vessel (BV). x2200. Bar = 25um. Inset xl60. Bar = lOOum. 185 186 Figure 42 Comparative longitudinal views of an intermediate (a) and red (b) fiber i n the normal diaphragm. Both xl7,500. Bar = l u m . Figure 42 a A n abundant transversely-oriented array of mitochondria is present wi th in the I band (I) of this fiber. Note the occasional mitochondrial columns (arrowheads) which are thin and span the A band (A). Sarcotubular elements show a we l l -developed and regular arrangement in A band and I band regions. Figure 42b Thickened mitochondrial columns are present i n the red fiber of the diaphragm. A well-developed SR network persists i n these fibers and shows intimate associations wi th mitochondria. 187 188 associated w i t h these mitochondrial columns was a delicate array of tubular elements comprising the sarcotubular system. Three-Dimensional infrastructure of Mdx Muscle EDL U p o n examinat ion of muscle fibers i n the mdx E D L , few obvious ultrastructural differences could be detected by H R S E M i n the majority of fibers when compared to those i n the normal E D L . Similar to their normal counterparts, muscle fibers i n the mdx E D L were separable into white, intermediate and red fibers based on the arrangement and density of mitochondria. Examples of a white fiber and a red fiber i n the mdx E D L are shown i n Figures 43 and 44, respectively. In white fibers (Fig. 43), mitochondria were thin and they partially encircled myofibrils at I band levels. These fibers also exhibited a dense SR network w i t h extensive junctional SR and tightly coalescing tubules wi th in fenestrated collars at the m i d - A band. Red fibers (Fig. 44), i n contrast, exhibited abundant mitochondrial columns containing internal cristae wi th a distinct lamellar arrangement. The SR of red fibers appeared normal and well-developed i n both A band and I band regions. T-tubules also exhibited both a normal morphology and a regular association wi th terminal cisternae of the SR. By H R S E M , the mitochondria and sarcotubular system of intermediate fibers i n the mdx E D L showed a compara t ive ly s imi lar ultrastructure to that observed in intermediate fibers of the normal E D L . Soleus Central nuclei were a common feature i n the majority of muscle fibers i n all three mdx muscles. Examples of 'centronucleation' are seen i n transverse (Fig. 45a) and longitudinal (Fig. 45b) views of intermediate fibers i n the mdx soleus. These nuclei were o v o i d i n shape, possessed a c lumped chromatin pattern, and 189 Figure 43 Long i tud ina l v i ew showing the arrangement of sarcotubular elements and mitochondria i n a white fiber of the mdx E D L . SR and T-tubules are regularly arranged and mitochondria are present as thin, transversely oriented profiles wi th in the I band. Note a rare column-forming mitochondrial branch (arrow) i n the A band. White circle indicates a triad. x36,000. Bar = l u m . 190 191 Figure 44 Longitudinally-fractured red fiber i n the mdx E D L seen at low (a), medium (b) and high (inset i n b) magnifications. Figure 44a Both the sarcotubular system and mitochondria are well-developed i n this fiber. x7200. Bar = 5 um. Figure 44b Enlargement of the boxed region i n a. A uniformly-size network of SR is seen i n the A band (A) and I band (I) regions. Large column-forming mitochondria occupy the intermyofibrillar regions and appear normal i n their morphology. x22,000. Bar = l u m . A h igh magnificat ion v iew of the internal lamellar cristae of two adjacent mitochondria is seen i n the inset. T-tubule (t). Inset x60,000. Bar = 0.5 um. 192 193 were typically arranged in a single, central, longitudinal row. Variable numbers of mitochondria were also commonly observed in close association with the outer nuclear membranes of these central nuclei. In addition, mitochondria and sarcotubular elements in intermediate fibers exhibited a regular and periodic arrangement comparable to that observed in intermediate fibers of the normal soleus. Red fibers of the mdx soleus contained a plethora of column-forming mitochondria both with pleomorphic shapes and with closely-packed internal cristae (Fig. 46). Similar to the red fibers of the normal soleus, a poorly-developed SR network was present with loosely-arranged fenestrated collars in mid-A band regions. Dilated cisternae of the SR were observed predominantly in the A band SR near junctional regions and at terminal cisternae. Bare, or non-junctional, T-tubules were commonly seen throughout the sarcoplasm of these fibers (Fig. 46). Necrotic lesions in mdx muscles were observed in varying degrees of severity in each muscle. Such regions appeared to be distributed in a non-specific pattern throughout the tissue. In a survey view of the mdx soleus (Fig. 47a), portions of the muscle can be seen to contain hollow, or 'moth-eaten,' fibers. These abnormal fibers were surrounded by a rich collection of mononuclear cells that are likely involved in the inflammatory response to these areas. In longitudinal fractures, degenerative fibers contained hollow cores with scanty amounts of disorganized membranous components in their interiors (Fig. 47b). Diaphragm The majority of fibers in the mdx diaphragm showed a remarkably normal and highly-developed array of both sarcotubular elements and mitochondria. Intermediate fibers (Fig. 48) contained both transverse and column-forming mitochondria that exhibited unusual complexity in their shapes (Fig. 48b). The SR 194 Figure 45 Low magnification transverse (a) and longitudinal (b) views of muscle fibers in the mdx soleus. Both x5700. Bar = 5um. Figure 45a Several intermediate fibers are seen, two of which exhibit centrally-placed nuclei (arrows). Note the fiber in the lower portion of the field which exhibits a vacant sarcoplasm (*). Figure 45b Longitudinal view of an intermediate fiber showing the typical alignment of nuclei (arrows) into a central row in the fiber core. Note the periodic and regular arrangement of both the sarcotubular system and mitochondria along the length of the fiber. 195 196 Figure 46 Longitudinal v iew of a red fiber in the mdx soleus showing large, pleomorphic, column-forming mitochondria and a delicate array of sarcotubular elements. Bare (or non-junctional) regions of T-tubules are indicated (arrows). Note the internal cristae i n mitochondria that have been fractured open. A band (A), I band (I). x20,000. Bar = l u m . 197 198 Figure 47 Examples of muscle fiber degeneration in the mdx soleus as seen i n transverse (a) and longitudinal (b) planes. Figure 47a L o w magnification survey v iew of the midbel ly region of the soleus showing healthy, centrally-nucleated fibers (to the left) and a necrotic region (to the right). Degenerative muscle fibers exhibit a hol low sarcoplasm, whi le the surrounding extracellular space exhibits a rich mononuclear cell infiltration. x270. Bar = lOOum. Figure 47b Longitudinal view of two degenerative fibers (*) showing their hollow cores and the disarray of both sarcotubular elements and mitochondria. Blood vessels (BV). x750. Bar = 50um. 199 Figure 48 Portions of a longitudinally-fractured intermediate fiber i n the mdx diaphragm seen at medium (a) and high (b) magnification. Figure 48a A subsarcolemmal nucleus contains two prominent nucleoli (*). The regular and periodic arrangement of the sarcotubular system and mitochondria can be seen in the sarcoplasm. x21,000. Bar = l u m . Figure 48b A highly anastomosing, uniformly-sized, tubular network of A band (A) and I band (I) SR is revealed at the level of the sarcomere. A triad is indicated at the A - I junction (circle). A n elongate, pleomorphic, canine-shaped mitochondrion exhibits a branch at the level of the I band. Note a small bridge of SR connecting the two terminal cisternae at the site of the A- I junction (arrow). x45,000. Bar = l u m . 201 202 was comprised of an evenly-sized network of tubules i n both A band and I band regions which exhibited an elaborate fenestrated collar at m i d - A band levels. The I band SR network was complex and highly anastomosing. Red fibers were typified by both large, co lumn-forming mitochondr ia and wel l -deve loped sarcotubular elements (Fig. 49a) i n addi t ion to dense accumulations of spherical-to-ovoid subsarcolemmal mitochondria (Fig. 49b). A noteable feature i n the mdx diaphragm was the presence of red fibers wi th an extremely h igh mitochondria l content (Figs. 50-51). In these fibers, the sarcoplasm was predominated by thick, elongate columns of mitochondria, some of which were observed to span up to 6 sarcomeres in length as a single unit (Fig. 50). The internal aspects of these mitochondria were characterized by a combination of both tubular and plate-like cristae (Fig. 51a). Sarcotubular elements were discrete and exhibited poorly-defined networks wi th in A band and I band regions, both of w h i c h were closely associated w i t h the outer surface of co lumn-forming mitochondria. L i p i d droplets and moderately distended portions of the A band and I band SR were also commonly observed in these fibers (Fig. 51b). Stereo Pairs To better appreciate the three-dimensional nature of the internal membrane systems of these muscle fibers, selected stereo pairs of muscle fibers from normal (Fig. 52) and mdx (Fig. 53) muscles have been included at the end of this section. A stereoviewer (Stereopticon 707, Taylor-Merchant Corp. , N Y ) is provided on the inside of the front cover for viewing of these stereo pairs. Instructions for its use are printed directly on the stereoviewer. 203 Figure 49 Red fiber i n the mdx diaphragm showing a port ion of its fiber core (a) and subsarcolemmal region (b). Both x33,000. Bar = l u m . Figure 49 a Mitochondrial columns are thick and exhibit extensive branching at the level of the I band. Two triads (circles) and a T-tubule (t) coursing over a mitochondrial column are indicated. A band (A), I band (I). Figure 49b A n accumulation of round-to-ovoid mitochondria is seen immediately beneath the sarcolemma (arrow) wi th intermingling elements of the SR. Several mitochondria have been fractured-open revealing their internal cristae. 204 Figure 50 Longitudinally-fractured red fiber i n the mdx diaphragm wi th an extremely high mitochondrial content. Thick, elongate mitochondrial columns span up to 6 sarcomeres i n length (*). A t higher magnification (below), a discrete sarcotubular system decorates the outer mitochondrial surfaces. A band (A), I band (I). xl0,000. Bar = lOum. Enlarged regions x24,000. Bar = l u m . 206 207 Figure 51 High magnification longitudinal views of red fibers in the mdx diaphragm. Figure 51a Combinations of plate-like (arrow) and tubular (*) cristae are seen in these column-forming mitochondria. x43/000. Bar = lum. Figure 51b Several mitochondria exhibit a prominent network of worm-like, tubular cristae. A lipid droplet (L) occupies an intermitochondrial space. Cisternal elements of the A band SR (*) and I band SR (arrowheads) are observed. Note the triad (circle) at the A-I junction. x57,000. Bar = lum. 208 Figure 52 Selected H R S E M stereo pairs of normal muscle fibers. Figure 52a L o w power H R S E M , E D L . Striations correspond to the sarcotubular system and mitochondria i n two longitudinally-fractured white fibers. x700. Figure 52b Transversely-fractured intermediate fiber i n the normal E D L showing the honecomb arrangement of the mitochondria and sarcotubular system around myofibrils (spaces). xl3,000. Figure 52c Red fiber i n the normal diaphragm, longitudinal v iew. A delicate sarcotubular system embraces both the column-forming and transversely-oriented mitochondria. xl l ,250. 210 Figure 53 Selected H R S E M stereo pairs of mdx muscle fibers. Figure 53a Red fiber i n the mdx soleus, longi tudina l v iew. Note the large vesicular mitochondria and the cage-like network of the sarcotubular system. xl3,000. Figure 53b Intermediate fiber i n the mdx diaphragm, longitudinal view. Note a mitochondrial branch at the level of the I band and the well-developed sarcotubular system. T-tubules are indicated (arrowheads). A band (A), I band (I). x26,000. Figure 53c Red fiber i n the mdx diaphragm, longitudinal view. Three large, fractured-open mitochondria exhibit tubular cristae. Note elements of the sarcotubular system to the right and a l i p id droplet in the center. x39,000. 212 213 D I S C U S S I O N The present H R S E M study extends the previous light microscopic (Chapter 1) and T E M (Chapter 2) observations by adding new information on the three-dimensional morphology of mitochondria and the sarcotubular system of skeletal muscle fibers i n both normal and mdx mice. Ultrastructural differences were noted between mitochondria and sarcotubular systems of white, intermediate and red muscle fibers i n three functionally diverse skeletal muscles ( E D L , soleus and diaphragm) of both normal and mdx mice. There are several advantages of using the A - O - D - O procedure (Tanaka and Mitsushima, 1984) for H R S E M v iewing of intracellular membranes. First, the selective extraction of the contractile proteins and other nonmembrane-bounded elements out of the sarcoplasm results i n a greater depth of field to be obtained in the fractured surface of the muscles. Secondly, large areas of the specimen can be quickly surveyed since sectioning is not required. Thirdly, fol lowing extraction of the cytosol w i t h dilute O s 0 4 , the three-dimensional arrangement of intracellular structures can be examined from several aspects (Lea et al., 1992). The chemical properties of O s 0 4 that allow it to act both as a fixative and an extraction agent are st i l l not fully understood. M u c h of the information on its action i n biological material has come from studies i n v o l v i n g either protein solutions or gels (Emerman and Behrman, 1982; Sjostrand, 1989). It has been generally accepted that O s 0 4 acts in its initial phases as a fixative or cross-linker of tissue proteins (Nielson and Griffith, 1979) and also as an oxidizer of specific fatty acids, such as olein (Porter and Kal lman, 1953). After long fixation times, however, a denaturation of proteins occurs. This has been demonstrated by the disappearance of an amorphous cytosolic matrix associated w i t h the formed bodies of the cytoplasm (Porter and Kal lman, 1953). Lea and coworkers (1992) have postulated 214 that the process of cytosol extraction with dilute O s 0 4 is based on oxidation of unsaturated lipids sandwiched between the two hydrophobic layers of exposed organelle membranes, and that the elemental form of osmium is deposited on the proteinaceous outer layers of the membranes. They predict that this osmium layer protects and preserves the structure of the membrane. Protein not in continuity with the l ipid is unprotected and undergoes denaturation and degradation with continued exposure to O s 0 4 . These breakdown products subsequently diffuse out of the cell during the extraction process. Whereas the A - O - D - O method provides detailed observation of membranous structure, an obvious disadvantage is the loss of morphological detail of protein structure and its interactions with the membranous elements of cells. Therefore, a major hurdle for future studies utilizing H R S E M will be to develop specific preparatory methods that both preserve and reveal these important membrane-cytoskeletal interactions within cells. H R S E M observations of the three basic muscle fiber types in the normal E D L revealed that mitochondria take on many different shapes and sizes. The highly branched nature of mitochondria in the fiber core of red fibers contrasted to those of the simpler, thinner and cylindrical-shaped mitochondria in white fibers. These variations in mitochondrial morphology suggest functional differences in energy utilization, resistance to fatigue, and contractile efficiency between these fiber types. Whereas red fibers of the E D L exhibited prominent and thickened columns of mitochondria along the length of myofibrils as well as accumulations of spherical-to-ovoid subsarcolemmal mitochondria, these features were not observed in white fibers. Intermediate fibers in the E D L were characterized by thin mitochondrial columns and fewer collections of subsarcolemmal mitochondria in comparison to those seen in the red fibers. A similar arrangement and distribution of mitochondria were observed in the intermediate and red fibers of the soleus and diaphragm muscles, although the 215 red fibers i n the diaphragm exhibited a higher content of mitochondria and were characterized by extremely thick columns that extended over several sarcomeres along the length of myofibrils. The concept of a 'mitochondrial reticulum' i n limb and diaphragm muscle fibers has been proposed previously by other investigators ut i l izing conventional and high-voltage T E M (Bakeeva et al., 1978, 1981; K i r k w o o d et al., 1986, 1987). Muscle fibers containing an abundance of mitochondria have been reported, by these investigators, to possess intermitochondrial junctions at sites where two mitochondria come together. These junctions have been generally described as contacts between the outer membranes of two mitochondrial branches. In addition, spaces between outer and inner membranes at these junctions have been reported to be filled wi th an osmiophilic substance (Bakeeva et al., 1978). A proposed hypothesis is that the 'mitochondrial ret iculum' serves as a system for transport of energy, oxygen and fatty acid residues along mitochondrial membranes over distances commensurate wi th the muscle fiber diameter (Bakeeva et al., 1978, 1981). These interconnections i n the mitochondrial system have been examined by introducing fluorescent probes which selectively accumulate i n the mitochondrial matrix of fibroblasts and cardiac myocytes and can be subsequently traced by microscopic techniques (Amchenkova et al., 1988) leading to the prediction that mitochondrial filaments or networks represent a united electrical system. In addition, mitochondrial networks have been shown to exhibit dynamic structural changes i n growing cells, w i t h tubular sections d iv id ing i n half, branching, and fusing to create a f luid tubular web (Bereiter-Hahn and Voth , 1994). Furthermore, alterations i n mitochondrial shape and distribution i n oocytes of Xenopus laevis have been s h o w n to be developmental ly programmed, w i t h characteristic morphologica l changes and migra t ion occurr ing at key stages of cellular differentiation (Mignotte et al., 1987; Barnett et al., 1996). Thus, it has become clear 216 that mitochondria are extremely dynamic organelles that possess the ability to modify their structure to suit the demands of the cell. In the present s tudy, i n d i v i d u a l mi tochondr i a exhib i ted elaborate configurations and their spatial distributions were characteristic for each fiber type. There was no evidence, however, that a completely interconnected system of mitochondria was present i n the muscle fibers (i.e. i n those fibers r ich i n mitochondria). Muscle mitochondria appeared, for the most part, as separate entities i n both the fiber core and subsarcolemmal regions, and only a few exhibited discrete extensions between these two areas of the fiber. It is predicted that their size and complexity relate pr imari ly to the overall content of mitochondria which , i n turn, is determined by the functional and energetic demands of the muscle fiber. A l though an interconnected network cannot be totally ruled out, the complex branching nature of muscle fiber mitochondria indicates that these organelles can span large regions of a fiber w h i c h l ike ly facilitates intracellular transfer of mitochondrial material and metabolites. Previous H R S E M studies have shown that both the internal structure as wel l as the external shape of mitochondria can vary dramatically i n cells of different mammalian tissues (Lea and Hollenberg, 1989; Lea et al., 1994). For example, i n cells of the adrenal cortex and liver of the rat, Lea and coworkers (1994) have shown that mitochondria are normally spherical-to-ovoid i n shape and contain predominantly tubular cristae. In contrast, the cells of b r o w n fat were reported to contain mitochondria w i t h internal cristae that exhibit a distinct shelf- or plate-like morphology. Whereas this group also briefly described the extensive branching nature of mitochondria i n skeletal muscle tissue of the rat, their study d id not include a comprehensive examination of mitochondria i n the various fiber types. Similar to the results of the present study, however, they reported a combination of tubular and plate-like cristae i n larger mitochondria (eg. the column-forming 217 mitochondria observed i n red fibers i n the present study) whereas smaller mitochondria contained mainly tubular cristae. The variabil i ty of mitochondrial morphology was further reinforced by observations of cultured human fibroblasts, in which small-diameter, thread-like mitochondria were seen to span distances of up to 46 um i n the cytoplasm (Lea et a l , 1994). It has now become evident from our observations and from those of others that mitochondria i n mammals show variations i n their structural organization and their inner cristae, as we l l as the topography of their exterior membranes. Functionally, white fibers of mammalian fast-twitch skeletal muscles are known to be rapidly contracting, fatiguable, and glycolytic (Dubowitz and Pearse, 1960; Kugelberg and Edstrom, 1968). They have been reported to contain a dense network of a narrow, uniformly-sized SR and a relative paucity of mitochondria. Moreover, it is we l l recognized that muscle fibers w i th faster activity cycles have more T-tubule and SR elements (Franz in i -Armst rong , 1973). In contrast, mammalian red fibers are known to be slower-contracting and fatigue-resistant wi th many mitochondria and a less-developed sarcotubular system (Ogata and Murata, 1969; Tomanek et a l , 1973). The three-dimensional ul t rastructural features of the SR i n whi te , intermediate and red fibers of fast-twitch muscles has been demonstrated by H R S E M in other mammalian species including rat (Ogata and Yamasaki, 1985b) and humans (Ogata and Yamasaki, 1997). In these studies, however, no significant differences in SR structure were described, although it was pointed out by these workers that small qualitative differences were present i n both the volume of the SR elements and their relationship to mitochondria. Our H R S E M results demonstrate more obvious differences i n the organization and spatial distribution of SR between muscle fibers of the E D L (a fast-twitch muscle) and muscle fibers of the soleus (a slow-twitch muscle). In addition to an overall lower content of SR i n the muscle fibers of the 218 soleus (Chapter 2), the structure of the fenestrated collar at m i d - A band regions showed a distinct variation between muscle fibers i n these two muscles. In the red fibers of the soleus, a poorly-developed SR network w i t h a wide variation in SR tubule diameters was observed. Moreover, i n contrast to the tightly-coalescing, tubular nature of the fenestrated collar i n the E D L muscle fibers, both intermediate and red fibers of the soleus exhibited wide SR fenestrae wi th in the A band regions. Similar differences have been noted i n H R S E M studies of the SR of frog and avian fast-twitch and slow-twitch fibers (Ogata and Yamasaki, 1993) although, unti l the present study, these differences between the fast-twitch and slow-twitch fibers have not been previously reported in mammals by H R S E M . It is generally accepted that mammalian skeletal muscle fibers have two T-tubules per sarcomere which are normally present at the A - I junctions. This is i n contrast to that seen i n cardiac muscle fibers and i n skeletal muscle fibers of amphibians where T-tubules occur at the level of the Z bands (Peachey, 1965; Forbes and V a n Nie l , 1988; Ogata and Yamasaki, 1993). The T-tubular system functions to transmit the surface membrane depolarization into the interior of the muscle fiber resulting, ultimately, i n the release of calcium ions from the SR (Huxley and Taylor, 1958; Sandow, 1965; Caputo, 1978). Observations i n the present study revealed a virtual absence of bare or non-junctional regions of the T-tubules i n fibers of the E D L and diaphragm. This was i n contrast to that seen in the red fibers of the soleus in which bare regions of T-tubules were often observed coursing over the column-forming mitochondria and between the myofibrils enroute to a neighbouring SR network. Thus, it is presumed that the lower proportion of junctional regions (i.e. triads) i n red fibers of the soleus is related to the low content of SR wi th in these fibers. Moreover, i n functional terms, the relatively high occurence of these bare regions of the T-tubules i n addi t ion to the low content of SR may represent 219 ultrastructural features that correlate wi th the slow-twitch properties associated wi th soleus muscle fibers. The diaphragm represents a highly specialized skeletal muscle designed for the continuous and repetitive activity of respiration. Al though several T E M reports have been pub l i shed on the ultrastructure of both mi tochondr ia and the sarcotubular system i n diaphragm muscle fibers (Gauthier and Padykula , 1966; Bakeeva et al., 1978; Gottschall, 1980; Ki la rsk i and Sjostrom, 1990), this is the first study to utilize the three-dimensional v iewing capabilities of H R S E M to examine their spatial arrangement i n cytosol-extracted muscle fibers. In the mouse, muscle fibers of the diaphragm exhibit a high oxidative capacity (Green et al., 1984) and possess a h igh content of histochemically identifiable 'red' fibers (see Chapter 1) which correlates wel l wi th the abundance and complexity of mitochondria observed i n these fibers by H R S E M . Comparat ive studies of mamalian diaphragms have revealed that small animal species typically exhibit small muscle fibers r ich i n mitochondria which are relatively fast and resistant to fatigue (Gauthier and Padykula , 1966; Green et al., 1984). Animals intermediate i n size, such as rabbit and rat wh ich have lower metabolic rates than smaller animals, have been shown to contain a heterogeneous mixture of fibers (Kilarski and Sjostrom, 1990). However , i n large animals which have even lower metabolic activity and slower breathing rates, large fibers w i th relatively low mitochondrial content predominate (Gauthier and Padykula, 1966). These observations have led to a general premise that oxidative capacity of muscle fibers is inversely related to body size (Green et al., 1984). In addi t ion to an abundance of mitochondria i n muscle fibers of the diaphragm, our H R S E M observations also revealed that these muscle fibers contain an elaborate sarcotubular system comparable to that observed i n muscle fibers of the fast-twitch E D L . The well-developed SR would provide more surface area and most 220 l ike ly corresponds to speed of contraction and relaxation times. Thus, the continuous and repetitive contractile activity of the diaphragm, coupled wi th the relatively high breathing rate of the mouse and the rapid speed of contraction of the diaphragm i n this animal (Crosfill and Widdicombe, 1961) conform wel l w i th the ultrastructural observations of organelle design i n these fibers. H R S E M observations of mdx muscle fibers i n the present study revealed few ultrastructural differences i n mitochondria and sarcotubular elements from those observed i n their normal counterparts. In al l three muscles examined, 'healthy' appearing muscle fibers exhibited a similar d is t r ibut ion and complexity of mitochondria i n all three fiber types. The highly branched nature of mitochondria in mdx muscle fibers was consistent w i th those observed i n normal muscle fibers. Their internal cristae appeared to be unaltered and exhibited a regular array of tubular and shelf-like configurations. M d x muscle fibers also displayed comparable sarcotubular systems to those observed i n the corresponding fiber types of normal muscles. One notable difference, however, was that the majority of 'healthy' mdx fibers contained central rows of nuclei wh ich are thought to be indicative of regeneration i n muscle fibers (Karpati et al., 1983; Engel and Banker, 1986; Anderson, 1991). Wi th in the mdx E D L , soleus and diaphragm, necrotic or degenerative fibers were dispersed throughout the muscle tissue either indiv idual ly or i n groups. By H R S E M , degenerative fibers had a 'moth-eaten' appearance and exhibited a hollowed-out sarcoplasm. Only scanty amounts of mitochondria and sarcotubular elements were present in these cells, although near the periphery of these abnormal fibers some formed elements could be recognized. In large necrotic regions, an increase i n extracellular matrix components was also noted. In addition, extensive infiltration of mononucleate cells characterized these regions. Precise identification of these mononucleate cells, however, was difficult by H R S E M since they were 221 rarely fractured open to reveal their internal cytoplasmic components and were surrounded by a dense meshwork of connective tissue. Three-d imens iona l morpho log ica l studies of mi tochondr i a and the sarcotubular system i n muscle fibers either i n dystrophic or experimental pathological conditions of muscle tissue are few i n number (Scales and Yasumura, 1982; Goto et al., 1990; Iwasaki and Suzuki , 1991; Kawahara et al., 1991). Scales and Yasumura (1982) were the first group to study the three-dimensional morphology of both the mitochondria and the sarcotubular system in thick sections of dystrophic chicken skeletal muscle. They used a special technique that involved the diffusion of electron-dense material (silver) to selectively enhance the contrast of the sarcotubular system. Their results indicated abnormalit ies at SR-T-tubule junctional regions in the diseased muscle, including the presence of narrow tubules of SR wh ich were often coiled around T-tubules instead of making the usual extended cisternal appositions. They concluded that their results explained the abnormalities they had observed earlier i n thin-sectioned material. They also illustrate the importance of this technique for the interpretation of the internal membrane networks of muscle tissue. In conclusion, the results of this H R S E M study confirm and extend our previous T E M observations of the E D L , soleus and diaphragm of normal and mdx mice (Chapter 2) by demonstrating differences i n the structure and content of mitochondria and the sarcotubular system between fiber types i n these three functionally-diverse muscles. It is particularly noteworthy that the advantages of H R S E M permit the mitochondria and the sarcotubular system to be revealed in relief and w i t h three-dimensional perspective, aspects not normally appreciated i n two-dimensional thin-sections by T E M . 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Use of a Cerium-Based Ultracytochemical Method for the Study of Ca lc ium- and Magnesium-Dependent Phosphatase Ac t iv i ty i n the Sarcoplasmic Reticulum. I N T R O D U C T I O N Calcium-Transporting Enzymes Intrinsic membrane proteins that regulate intracellular levels of calcium (Ca 2 + ) ions have become of paramount interest because of the critical involvement of this ion i n intracellular signalling. In skeletal muscle, a sarcoplasmic increase of C a 2 + activates the process of muscle fiber contraction, whereas relaxation of the fiber is dependent on the removal of C a 2 + from the sarcoplasm, either into internal storage sites such as the sarcoplasmic re t iculum (SR) and mitochondria , or extracellularly. The non-uniform distribution of C a 2 + is maintained by specialized Ca 2 +-transporting systems embedded wi th in the sarcolemma and the membranes of the SR and mitochondria . These systems include the N a + - C a 2 + exchange mechanism, the ATP-dependent C a 2 + pump of the sarcolemma, and the highly-efficient Ca 2 + -activated adenosine triphosphatase pump (Ca 2 + -ATPase) of the SR. Each exhibits a different affinity to C a 2 + , total transport capacity, electrogenecity, and sensitivity to modulating agents (Carafoli, 1988). Muscle relaxation is largely dependent on the activity of the SR-Ca 2 + -ATPase (Carafoli, 1988) wh ich can pump C a 2 + ions from the sarcoplasm into the lumen of the SR against concentration gradients of up to three orders of magnitude (MacLennan and Campbell , 1979). This ATPase has been shown to constitute up to 85% of the total membranous SR protein of some skeletal muscles (Martonosi and Beeler, 1983). It has been isolated as a single polypeptide of 115 k D (Brandl et a l , 1988) and is asymmetrically distributed i n the SR membrane (Deamer and Baskin, 229 1969; Saito et al., 1978) with much of its mass exposed on the cytoplasmic surface, where it can be visualized by negative staining of the surface particles (Martonosi, 1968; Ikemoto, 1982). A reaction scheme for the translocation of Ca 2 + across the SR membrane has been proposed by de Meis and Vianna (1979) and is generally accepted as the mechanism of active Ca 2 + transport associated with the SR (see below). 2Ca 2 + ( U Co. Co' ATP _ L (2) Ca Ca : E *A T P ADP J _ (3) C ° : E ~ P (8) (4) *F — 7 —*P -P C 2 J t-° ( 6 ) 2 Y * E . - P pi HOH (5) TI Co' * - 2 2Ca Reaction scheme for the Ca 2 + + Mg2 + - activated ATPase (de Meis & Vianna, 1979) The cycle begins with the Ca2+-dependent transfer of the terminal phosphate of ATP to the pump in the E l conformation on the sarcoplasmic side of the membrane (steps 1-3) (Katz and Blostein, 1975; Martonosi and Beeler, 1983). Coupled to ATP hydrolysis and the loss of ADP, a phosphorylation of an aspartyl group in the active site drives a Mg2+-promoted conformational transition from the E l to the E2 state (step 4) and, in the process, translocates two Ca 2 + ions from the sarcoplasmic side to the luminal side of the SR membrane (step 5). At the end of the reaction cycle, the dephosphorylated E2 conformer reverts back to the starting E l conformation (steps 6-8). The enzymatic catalysis by Ca 2 +-ATPase is a fully 230 reversible, coupled vectorial reaction that transports C a 2 + ions w i t h a stoichiometry to A T P of 2:1 (de Meis and Vianna, 1979). The E l conformation of the ATPase has been shown to be stabilized by saturation of the high-affinity C a 2 + - b i n d i n g site of the enzyme w i t h C a 2 + or lanthanides (Dux et al., 1985). Removal of C a 2 + from the enzyme w i t h ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and binding of orthovanadate stabilize the E2 conformation (Pick, 1982; Pick and Karl ish, 1982). It is generally assumed that vanadate inhibits Ca 2 + -ATPase by b inding to the aspartyl residue that forms the phosphorylated intermediate at the active site (Carafoli, 1991). M u c h has been learned about the three-dimensional structure of the C a 2 + -ATPase through crystal l ization studies of the enzyme i n SR membranes and reconstituted ATPase vesicles i n the presence of vanadate and lanthanide or C a 2 + ions (Franzini -Armstrong et al . , 1986). It has been demonstrated that two-dimensional crystalline arrays of Ca 2 + -ATPase molecules develop i n SR vesicles exposed to calcium or lanthanide ions at a moderately alkaline p H (Dux et al., 1985). These C a 2 + - or lanthanide-induced crystals are bel ieved to represent the E l conformation of the enzyme, and their crystal form is clearly different from the vanadate induced crystalline pattern (Taylor et al., 1984). In the lanthanide-induced E l state, electron microscopic analysis reveals obliquely oriented rows of particles corresponding to ind iv idua l Ca 2 + -ATPase molecules (Dux et a l , 1985). Computer analysis of the crystalline arrays shows that they are composed of rows of monomeric units, each unit measuring approximately 6 nm i n diameter (Dux et a l , 1985). In the presence of vanadate, the E2 conformation of the Ca 2 + -ATPase enzyme can be visualized i n SR vesicles (Dux and Martonosi, 1983). Filtered image analysis of vanadate-treated SR vesicles demonstrates that the vesicle surfaces are covered by helically arranged, long rows of paired units existing as dimers (Buhle et al., 1983; Castellani and Harkwicke, 1983; Taylor et a l , 1984). 231 Proposed structural models for these ATPases on the basis of sequence data suggest that these transport proteins are organized into functional domains wi th discrete domains for nucleotide and Ca 2 + -b ind ing and a transduction domain responsible for transmitting conformational changes between domains, coupling A T P hydrolysis to C a 2 + translocation (MacLennan et al., 1985). The alternation between the two distinct conformations of Ca 2 + -ATPase during the transport cycle is supported by changes in the affinity of the enzyme for C a 2 + , M g 2 + , A T P , and A D P (Martonosi , 1982). There are no net changes i n the polypept ide backbone conformation on t ransi t ion between the E l and E2 states; therefore, the conformational change is l ikely to involve either a hinge-type or relative sl iding motion of domains (Csermely et al., 1987). Calcium Balance and Muscular Dystrophy When compared to the C a 2 + concentrations i n normal human and mouse skeletal muscle, intracellular resting free C a 2 + concentrations i n myotubes of Duchenne muscular dystrophy ( D M D ) and mdx mouse or ig in are significantly higher (Fong et al . , 1990). One hypothesis suggests that the increased open probability of C a 2 + leak channels contributes to the elevated free intracellular C a 2 + concentration i n D M D and mdx myotubes. Ut i l i z ing the calcium indicator fura-2, Turner and coworkers (1991) demonstrated that both mdx myotubes and muscle fibers are less able to regulate intracellular C a 2 + levels i n the region near the sarcolemma. Moreover, C a 2 + transport or gating-mechanism defects have also been described (Head, 1993; Menke and Jockusch, 1991) which, i n turn, may contribute to the hypercontractility and subsequent destruction of the diseased fibers. Specific activity of C a 2 + - A T P a s e and the abil i ty of the SR to regulate intracellular levels of C a 2 + i n dystrophic muscle fibers have been considered to be a factor i n D M D muscle fiber necrosis and have been investigated by a number of 232 researchers (Martonosi, 1967; Nagy and Samaha, 1984a, b, c, 1986). SR membrane changes observed i n D M D skeletal muscle fibers have been attributed to a significantly higher protease activity in the diseased muscle and, i n particular, those affecting the protein constituents of the Ca 2 + -ATPase pump (Nagy and Samaha, 1984a, b, c, 1986). The Ca 2 +-activated neutral proteases, named calpains, hydrolyze SR proteins and other components of the muscle fiber. They have been proposed to be a major factor leading to the degradation of intracellular proteins (Ishiura et al., 1979; Kameyama and Etlinger, 1979; Sugita et al., 1980) and membrane systems (Nagy et al., 1986), the degenerative processes observed in D M D skeletal muscle fibers. A proposed hypothesis for these degenerative processes, therefore, is that the intracellular C a 2 + regulatory mechanism is altered i n D M D and mdx muscle fibers. Al though activity of the Ca 2 + -ATPase has been investigated in vitro by others using various isolation methods (Nagy and Samaha, 1984a, b, c, 1986; Land i et al., 1986), there are currently no studies that have examined its in situ morphological and functional status using cytochemical procedures. For this reason the fol lowing study was undertaken to determine its topographical localization i n skeletal muscle fibers and to elucidate possible differences i n its activity i n the mdx-dystrophic condition. The methods chosen for this study were based on their abilities to reveal both fine structural detail and functional SR enzyme activity; these are discussed i n the following section. Cerium-Based Ultracytochemistry To date, studies using enzyme ultracytochemical techniques for the in situ localization of Ca 2 + -ATPase activity have been difficult to interpret due to non-specific precipitation of reaction product especially when lead is used as the capture agent (Gauthier, 1967; Zinchuk and Bulavka, 1992). The cerium-based method of enzyme detection coupled wi th the use of para-nitrophenylphosphate (pNPP) as 233 substrate is a recently developed technique that permits the ultrastructural demonstration of phosphatase activity (Seguchi, 1993). It has several advantages over the established lead-based method inc lud ing a finer reaction product precipitate and reduced non-specific staining (Robinson and Karnovsky, 1983a, b; Kobayashi et al., 1987). Exis t ing as a trace element i n the body, the lanthanide cerium exhibits numerous potential applications as an ion-capturing agent for detection of A T P -driven enzymatic reactions. Its advantages have been recognized i n histochemical applications (Veenhuis et al., 1980; Robinson and Karnovsky, 1983a, b; Kobayashi et al., 1987) and have made it a method of choice over the lead-based method for cytochemical detection of free orthophosphate (Veenhuis et al., 1980) and hydrogen peroxide (Briggs et al., 1975) due to the extremely low solubil i ty of cerium III-phosphate and cerium IV-perhydroxide i n hydrophi l ic incubation media (Feigl, 1958; Moller , 1963). Theoretically, the activity of all oxidases and phosphatases can be revealed histochemically w i t h the use of cerium. Several studies have confirmed by x-ray microanalysis that a cerium-containing reaction product is generated under physiological conditions i n tissue sections at the sites of enzyme activity (Karnovsky et al., 1981; Ohno et al., 1982). It is this high specificity and affinity properties of cerium that make it a reliable marker for phosphatases and oxidases. It is also a procedure that is vir tual ly free of diffusion and affinity artifacts, exhibiting fine granularity of the reaction product (Kobayashi et al., 1987; Noorden and Frederiks, 1993; Halbhuber et al., 1994). In addition, the methods are highly reproducible and cerium has a minimal inhibitory effect on at least a number of enzymes (Robinson and Karnovsky, 1983a, b; Angermueller and Fahimi, 1984; Hulstaert et al., 1989; V a n Goor et al. , 1989; L u o et al., 1990). Unl ike the lead-based techniques, cerium sensitivity is existent throughout a wide range of pH's (Luo et al., 1990) making it 234 suitable for demonstration of acid, neutral and alkaline phosphatases as well as for oxidases. At the electron microscopic level, sites of phosphatase activity can be demonstrated by the precipitation of cerium Ill-phosphate (Hulstaert et al., 1983; Robinson and Karnovsky, 1983a). The reaction that leads to the formation of this precipitate is shown below: phosphatases Ce 3 + (aq)+ substrate-P04 > CeIII P0 4 (s) + substrate residue Orthophosphate is split from the substrate through the activity of phosphatases. The liberated orthophosphate then binds to the capturing ion (cerium) to form a water-insoluble reaction product, cerium Ill-phosphate, which can then be visualized by electron microscopy. The proposed reaction scheme at the SR membrane is seen below. 235 The use of A T P for detection of phosphatase activi ty at the electron microscopic level has been attempted by several workers (Novikoff, 1970; Rosenthal et al., 1970). Its use, however, has been limited due to the abundance of non-specific precipitation i n tissue preparations. The magnesium salt of p N P P has been shown to be an effective substrate for use i n phosphatase research for a variety of phosphatases. It is noteworthy that p N P P has been used w i t h success as a pseudosubstrate i n place of A T P to support active transport of C a 2 + i n SR vesicles due to similarities i n its catalytic site wi th that of A T P (Rossi et al., 1979). Moreover, it has proven by these workers to be an excellent substrate for use i n phosphatase research since its hydrolysis yields the yel low end-product, p a m - n i t r o p h e n o l (pnitrophenol), which can be measured spectrophotometrically (see below). ATPase /mitrophenylphosphate > pnitrophenol (yellow) + P 0 4 A n obvious advantage of its use, therefore, is to allow for the quantitative determination of enzymatic activity under cytochemical conditions (Rossi et al., 1979; Kobayashi et al., 1987; Seguchi, 1993). P 0 4 , on the other hand, serves as a target for a capture ion such as cerium to form an insoluble electron dense precipitate as mentioned previously. Establishing optimal conditions for maintaining preservation of both tissue enzyme activity and structural details is essential i n this technique and should be opt imized when either modifying or developing protocols. ATPase activity, i n general, is sensitive to fixation wi th glutaraldehyde (Moses et al., 1966; Ernst and Philpott, 1970) and shows inhibitory effects at those concentrations used for routine morphological procedures i n electron microscopy. The use of paraformaldehyde as the primary fixative has been shown to exert a substantially lower inhibi t ion of 236 ATPase activity (Kobayashi et al . , 1987) whi le at the same time preserving ultrastructural details. It has, therefore, become the fixative of choice for enzyme ultracytochemistry. The primary aims of this portion of the study are to first develop and test a method that enables the ultracytochemical demonstration and functional analysis of SR Ca 2 + -ATPase activity i n skeletal muscle tissue. Secondly, to utilize this protocol to test the hypothesis that the activity of Ca 2 + -ATPase i n the SR is altered i n mdx mouse skeletal muscle. Us ing these methods, both the topographical and functional nature of this membrane-embedded pump enzyme w i l l be assessed i n skeletal muscle fibers of the age-matched adult normal and mdx-dystrophic mice. 237 M A T E R I A L S A N D M E T H O D S Animals A d u l t (25-32 wk) genetically-dystrophic mice of the C57Bl/10ScSnMdx strain (n=3) and their normal C57Bl/10ScSn counterparts (n=5) were used i n this study. Original breeding pairs of both the normal and dystrophic strains were obtained from Jackson Laboratories, Bar Harbour, M E . They were subsequently maintained i n the mouse colony i n the Department of Anatomy at the Univers i ty of Brit ish Columbia. Two of the five normal mice were used i n preliminary experiments to test the effects of fixation, cerium and C a 2 + concentrations in the incubation media. Cytochemistry For this study, the gastrocnemius muscle was chosen due to its relatively large size and ease of tissue microslicing, a necessary procedure i n cytochemistry to improve accessibility and penetration of the incubation media into the tissue. The large size of this muscle also allowed for sufficient amounts of tissue to be collected and ut i l ized for comparison of incubation media. Halothane-anesthetized mice were perfused via the left ventricle wi th 2% paraformaldehyde i n 0.1 M cacodylate buffer ( p H 7.2) w i th 5% sucrose for 5 min. The gastrocnemius muscle was then removed from both legs and immersion fixed for 40 min i n fresh fixative on ice. Fol lowing a wash i n the same buffer, the tissue was adhered to a plastic block wi th glue and microsliced into 50-um-thick sections (Microslicer, Dosaka E M Co. , Kyoto, Japan). The sections were then transferred to a 50 m M Tricine buffer w i t h 5% sucrose on ice and indigenous free C a 2 + was removed from the tissue overnight on a reciprocal shaker w i t h increasing concentrations of ethylene glycol-bis(fi-amino-ethyl ether) N,N,N,N'-tetraacetic acid (EGTA) up to 0.5 m M (12 hr). To determine the optimal conditions for cytochemical detection of enzyme activity, biochemical experiments were performed wi th microsliced sections of fixed 238 tissues at p H 7.2. After removal of excess moisture from the tissue wi th filter paper, 10 mg of tissue was weighed out on a scale and incubated for 10-30 m i n on a reciprocal shaking incubator at 37°C i n 10 m l of a Tricine buffered medium (pH 7.2) containing the following: 100 m M potassium chloride, KC1 *0-5 m M cerium chloride, C e C l 3 (capture agent) 5 m M magnesium sulphate, M g S 0 4 0.5 m M E G T A 0.00015% Triton X-100 2 m M ouabain (inhibitor of Na +-K+ ATPase activity) 2 m M levamisole (inhibitor of alkaline phosphatase activity) 2 m M pflra-nitrophenylphosphate (pNPP) (magnesium salt) as substrate *0-5 m M calcium chloride, C a C l 2 . * A range of concentrations was tested. After incubation, the tissues were removed from the medium and placed i n Tricine buffer on ice to await further processing for T E M . A small sample of the incubation med ium was then wi thdrawn and the absorbance of p n i t r o p h e n o l produced by the hydrolysis of p N P P was measured at 405 nm i n a Hitachi 220A spectrophotometer (Hitachi Ltd . , Tokyo, Japan). Preparations of various reaction media were made to determine the effect of cerium ions and C a 2 + ions on enzyme activity. Concentrations of 0, 1, 2, and 5 m M C e C l 3 were tested i n a set of experiments to determine the time and dose dependence of cerium ions on enzyme activity. Free C a 2 + concentrations for optimal enzyme stimulation were determined by testing 0, 0.001, 0.01, 0.1,1.0, and 5.0 m M of C a C l 2 i n the incubation media. For controls, substrate-free incubation, high free C a 2 + incubation (5.0 m M ) , and addition of 2.0 m M of the Ca 2 + -ATPase inhibitor, thapsigargin (Lytton et al., 239 1991; Wi tcome et a l . , 1992) to the incubat ion m e d i u m were performed. Glutaraldehyde was not used i n this procedure since it was determined to have a strong inhibitory effect on enzyme activity. T E M Processing Following incubation procedures, specimens were rinsed i n cold tricine buffer and then transferred to 0.1 M cacodylate buffer containing 2% glutaraldehydye and paraformaldehyde. The samples were then washed i n cacodylate buffer at 4°C ( 3 x 1 0 min) and some were fixed w i t h buffered 1% O s 0 4 for 2 hr at 4°C to increase membrane contrast under T E M . They were washed i n distilled water (3 x 10 min), and stained wi th 1% aqueous uranyl acetate at room temperature for 1 hr. After a wash in distilled water (3 x 10 min), specimens were dehydrated through a graded series of ethanols followed by propylene oxide (3 x 15 min), and embedded in Spurr's epoxy resin (Spurr, 1969). Ul t ra thin sections (60-90 nm) were cut on a Reichert Ultracut O m U 4 ultramicrotome (Reichert-Jung, Vienna , Austr ia) and picked up on copper grids. To enhance structural contrast i n sections, some grids were stained wi th uranyl acetate and lead citrate (5 m in each). Observations were made w i t h a Hi tachi H-700 transmission electron microscope (Hitachi , Tokyo) operated at 75 k V . Reaction product i n ultrathin sections was examined by X-ray microanalysis on a JEM-1200EX (JEOL, Tachikawa, Japan) to confirm the presence of cerium i n the reaction product. 240 R E S U L T S Cytochemical spectrophotometric experiments performed on gastrocnemius muscles of normal mice revealed a concentration-dependent sensitivity of C a 2 + -Mg 2 + /?NPPase activity to cerium ions (Fig. 54a). Accumulat ion of /mitrophenol per mg of tissue was calculated using 0, 1, 2, and 5 m M of C e C l 3 i n the incubation medium. Inhibitors of the N a + - K + - A T P a s e p u m p (ouabain) and alkal ine phosphatase (levamisole) were always added to the medium i n these experiments to prevent their activities. A t high cerium concentrations (5mM), C a 2 + - M g 2 + pNPPase activity was reduced up to 64% at 30 min incubation time. The relationship of cerium ion concentration to C a 2 + - M g 2 + pNPPase activity was, therefore, both a dose-and time-dependent relationship. For ultracytochemical detection, a cer ium concentration of 2 m M and an incubation time of 30 m i n were chosen for demonstration of reaction product in the tissue since it provided a balance between detectability by T E M (i.e. accumulation of reaction product) and minimal inhibition of enzyme activity. The effect of free C a 2 + concentrations at quantities of 0, 0.001, 0.01, 0.1, 1.0, and 5.0 m M i n the incubation med ium was tested to determine opt imal enzyme activation over a 30 m i n period. A series of three separate experiments revealed maximal C a 2 + - M g 2 + pNPPase activity at free C a 2 + concentrations i n the range of 0.001 and 0.01 m M (Fig. 54b). L o w (<0.001 m M ) and high (>1 m M ) C a 2 + concentrations exhibited substantially reduced activity. A C a 2 + concentration of 0.01 m M was subsequently used i n the full medium to maximally stimulate C a 2 + - M g 2 + pNPPase activity. By T E M , ultrathin sections of the resin-embedded and stained tissue appeared relatively well-preserved and free from non-specific staining artifact (Fig. 55a). A t h igh magnification, sarcomeres, myofilaments, mitochondria and sarcotubular 241 Figure 54 Effect of cerium (a) and C a 2 + (b) concentrations on the activity of C a 2 + - M g 2 + pNPPase i n the normal mouse gastrocnemius. Hydrolysis of p N P P to pnitrophenol i n the incubation med ium was determined spectrophotometrically and represented i n p m o l e / m g / m i n . Ouabain and levamisole were always added to the assay medium. A cerium ion concentration of 2 m M was used for electron microscopic detection of enzyme activity. In b, specimens were incubated for 30 m i n i n incubation media wi th either 0.001, 0.01, 0.1, or 5.0 m M of free C a 2 + . Opt imal enzyme activation was obtained wi th free C a 2 + concentrations i n the micromolar range. Data were averaged from 3 separate experiments. 242 243 elements were easily distinguished and showed a regular arrangement (Fig. 55b). In addition, the SR, T-tubules, triadic couplings, and sarcolemma remained intact and were visibly distinguishable i n sections of the muscle fibers. In longitudinal section, muscle fibers contained varying amounts of electron-dense reaction product at the SR membranes depending on the posit ion of these membranes relative to the sarcomere. W i t h i n the sarcomere, white fibers of the normal gastrocnemius exhibited a h igh accumulat ion of reaction product at terminal cisternae on either side of the centrally located T-tubule (Fig. 55b). This dense staining was typically localized to the region of the SR immediately adjacent to the T-tubule and, i n some cases, appeared to be present w i th in the terminal cisternae. The fenestrated collar region of the SR exhibited relatively moderate staining which was typified by small accumulations of reaction product wi th in the centralized H-zone region of the sarcomere. Between the H zone and the terminal cisternae, the longitudinal portions of the SR displayed the least amount of activity i n comparison to other regions of the SR network (Fig. 55b). Whi le the I band SR was variable i n its staining, dense accumulations were always present at their junctional regions wi th the T-tubules. Periodic densities similar to those observed i n the m i d - A band SR were present in regions of the Z-line. The composition of the reaction product was analyzed by x-ray microanalysis by focussing the beam on a densely-stained portion of the SR membrane. Two recordings, one on a stained port ion of the SR membrane and the other on an unstained port ion of the SR membrane, are seen i n Figure 56. A h igh level of cerium was detected wi th in the reaction product (Fig. 56a) whereas the unstained region of the SR contained no detectable levels of cerium (Fig. 56b). In addition to SR-specific staining wi th in muscle fibers, there was prominent electron-dense reaction product associated w i t h the perinuclear cistern of the 244 Figure 55 L o w (a) and high (b) magnification views of the same white muscle fiber i n the normal mouse gastrocnemius showing the ultracytochemical localization of C a 2 + -M g 2 + pNPPase activity. Electron-dense deposits are present at junctional regions of the SR terminal cisternae (arrows). Moderate staining is seen at m i d - A band regions (*) and I band regions of the SR. Two triads are delineated by circles. Relatively little staining is seen at the sarcolemma above. Mitochondrion (Mi) , Z line (Z). a, x!2,000. Bar = 1 um. b, x54,000. Bar = 1 um. 245 246 Figure 56 Two recordings by x-ray microanalysis of an unstained (a) and stained (b) portion of the SR membrane i n a muscle fiber of the normal mouse gastrocnemius. Cer ium (Ce) was detected i n reaction product at the SR membrane (*). Oxygen (O), Copper (Cu), Osmium (Os), Chloride (Cl), Counts per second (cps), Kiloelectron volts (keV). 247 Figure 57 Longi tudinal v iew of a subsarcolemmal nucleus i n a white fiber of the normal gastrocnemius stained for C a 2 + - M g 2 + pNPPase. Note electron-dense reaction product in the perinuclear cistern of the nuclear envelope and at the terminal cisternae of the SR (arrows) i n the triads. x24,000. Bar = 1 um. 249 250 nuclear envelope (Fig. 57). This presented as periodic electron densities which encompassed the perimeter of the entire nucleus. Red fibers of the normal gastrocnemius, distinguished by their high content of subsarcolemmal and large column-forming mitochondria, exhibited relatively lower amounts of reaction product at SR membranes (Figs. 58a, b). It should be noted, however, that this was a qualitative assessment since it is v i r tua l ly impossible to obtain reliable quantitative density measurements of SR staining levels. The localization of reaction product was also primari ly observed at triads i n junctional regions of the SR on either side of the T-tubules. In addition, a moderate level of reaction product was also detected i n A band and I band SR tubules. Control experiments i n which tissue sections were incubated i n a substrate-free medium were consistently devoid of reaction product. These sections were always examined prior to contrast staining wi th lead citrate to eliminate any possible contamination by this stain. To determine the specificity of the incubation medium for SR C a 2 + - M g 2 + pNPPase, thapsigargin (2 m M ) , a known inhibitor of the SR C a 2 + -ATPase pump, was added to the reaction medium prior to incubation. Al though inhibi t ion was incomplete, a visible reduction i n staining was noted i n the SR. Partial inhibi t ion of this activity was also evident i n the spectrophotometrically-determined measurement of the conversion of p N P P to pni t rophenol i n the incubation medium (Fig. 59). Three repeat experiments were performed to compare the activity of the SR C a 2 + - M g 2 + pNPPase i n the gastrocnemius between normal and mdx mice. Equal amounts of tissue (10 mg wet weight) were incubated i n three different incubation media that included: 1) a full medium wi th al l ingredients for maximal enzyme stimulation, 2) a full medium containing 2 m M of thapsigargin and 3) a substrate free medium. After a 30 m i n incubation time, the conversion of p N N P to 251 Figure 58 Longi tudinal (a) and transverse (b) views of a red fiber i n the normal mouse gastrocnemius stained for C a 2 + - M g 2 + pNPPase. Reaction product is localized in the terminal cisternae and wi th in A band and I band regions of the SR. Vir tual ly no staining is seen at the sarcolemmal membrane i n the upper right. A transverse section of several myofibrils at the A band of the sarcomere (b) reveals small tubular profiles of the SR wi th a fine electron dense reaction product at their membranes. Mitochondrion (Mi), a, x20,000. Bar = 1 um. b, x60,000. Bar = 1 um. 252 253 pnitrophenol was measured spectrophotometrically i n each incubation medium. Absorbance values of the full incubation media revealed a significantly higher (25%, p<0.05) C a 2 + - M g 2 + pNPPase activity i n mdx muscle in comparison to normal muscle when incubated i n the ful l medium. Thapsigargin inhibi ted the activity by approximately 20% i n both normal and mdx tissue samples, whereas the substrate-free media exhibited negligible amounts of absorbance (Fig. 59). By T E M , mdx muscle fibers appeared to exhibit a qualitatively higher accumulation of reaction product at the SR membranes of white and red (Fig. 60) fiber types. Localization of reaction product was similar to that observed i n muscle fibers of normal tisssue i n terms of its location at terminal cisternae and i n the A band and I band portions of the SR. 254 Figure 59 Spectrophotometric comparison of the conversion of pNPP to /mitrophenol in reaction media containing either normal or mdx gastrocnemius tissue. Equal amounts of tissue were incubated in each medium for 30 min. A significant increase in activity was observed in the mdx tissue when compared to normal samples (p<0.05). Note a reduction in activity upon addition of the C a 2 + - A T P a s e inhibitor, thapsigargin, to the medium and negligible activity in substrate-free incubation media. Ouabain and levamisole were always added to the media. Error bars indicate standard deviation from 3 separate experiments. Significant differences (p<0.05) are indicated with an asterisk. 255 256 Figure 60 C a 2 + - M g 2 + pNPPase reactivity in a longitudinal section of a red muscle fiber from the mdx gastrocnemius. A h igh amount of reaction product is seen at the SR membranes. Note the large column-forming mitochondria between myofibrils (Mi) and the sarcolemma (upper right). x28,725. Bar = 1 Lim. 257 258 D I S C U S S I O N In this study, the ouabain-insensitive, C a 2 + - M g 2 + pNPPase activity i n normal and mdx skeletal muscle fibers was revealed by the cerium-based method of detection at physiological p H . Whereas the original method of Wachstein and Meisel (1957) which utilizes A T P as substrate and lead as the capture agent has been undertaken previously by several investigators (Gauthier, 1967; Giacomell i et al., 1967; Novikoff, 1970; Rosenthal et a l , 1970; Khan et al., 1975; Zinchuk and Bulavka, 1992), it was not uti l ized in this study due to several artifactual drawbacks including non-enzymatic, lead-catalyzed hydrolys is of A T P under reaction conditions (Novikoff, 1970; Rosenthal et al., 1970) and the presence of nucleotides i n the reaction product (Tice, 1969; Rosenthal et al., 1970). Instead of using A T P as substrate and lead as the capture agent, p N P P and cerium were u t i l i zed to avoid the complications endured wi th the previously attempted methods. To date, this is the first study to show C a 2 + - M g 2 + pNPPase activity in situ by cerium-based ultracytochemistry at the SR membranes of normal and mdx skeletal muscle fibers. Opt imal enzyme activity measured by the conversion of p N P P to pnitrophenol was obtained wi th free C a 2 + concentrations i n the micromolar range. This is i n general agreement wi th biochemical studies on microsomal SR vesicles of mammals i n w h i c h maximal s t imulat ion was obtained w i t h s imilar values (Martonosi, 1986). In the present study, activity was reduced upon addition of 2 m M thapsigargin (Lytton et al., 1991; Witcome et al., 1992) to the medium, indicating the specificity of the reaction medium for SR C a 2 + - M g 2 + ATPase. It was also determined that cerium has an inhibitory effect on enzyme activity wh ich increased linearly wi th increasing concentrations i n the incubation medium. To obtain a visually-dense reaction product under T E M , however, a 2 m M concentration of cerium chloride i n the medium was used for this study. 259 Localizat ion of enzymatic activity is i n general agreement wi th previously pub l i shed immunocy tochemica l (Jorgensen et a l . , 1979) and i m m u n o g o l d (Jorgensen et al., 1982; Dulhunty et al., 1987, 1993) studies that have localized C a 2 + -M g 2 + ATPase to the fenestrated collar regions and terminal cisternae of the SR in mammalian and amphibian skeletal muscle fibers. In skeletal muscle, most of the C a 2 + influx into the sarcoplasm for the stimulation of contraction is believed to come from the terminal cisternae and also from the longitudinal network of the SR (Winegrad, 1970). In functional terms, it wou ld be advantageous if these two C a 2 + -release sites were to possess efficient and speci f ica l ly- local ized p u m p i n g mechanisms for restoration of C a 2 + gradients. The results of the present study provide ultracytochemical evidence that C a 2 + -M g 2 + pNPPase activity is localized to distinct regions of the SR at different levels of the sarcomere. Moderate activity was observed i n the m i d - A band and I band SR whereas high activity was seen at the terminal cisternae. Vir tual ly no staining was associated w i t h either mitochondrial membranes, T-tubules, or the sarcolemma. These findings support the existence of topographical differences i n enzyme distribution wi th in the membrane of the SR. In addition, the presence of enzyme activity at the the nuclear envelope was also evident i n skeletal muscle fibers of both normal and mdx muscles. Nuclear envelope C a 2 + - M g 2 + ATPase activity has been reported i n several mononucleated cells including macrophages, thymic cells and lymphocytes (Mughal et al., 1989); however, its activity at nuclear membranes i n skeletal muscle tissue has not previously been confirmed. Evidence from this study supports the notion that both the SR and nuclear envelope i n skeletal muscle fibers share common C a 2 + - M g 2 + pNPPase activities and that these membranes are most l ikely continuous wi th each other. ATP-dependent, Ca 2 +-transporting mechanisms of striated muscle cells have been identified both i n the sarcolemma and i n intracellular organelles, and they can 260 be further distinguished by different functional properties such as sensitivities to modulating agents, transport capacity, and affinity for C a 2 + (Carafoli, 1988; Grover and Khan, 1992). For instance, i n skeletal muscle, the steady-state phosphorylation level i n the sarcolemmal C a 2 + - M g 2 + ATPase is usually much lower than i n the C a 2 + -M g 2 + ATPase of SR suggesting that the ratio between the rates of formation and b reakdown of the phosphoryla ted intermediate are less favorable i n the sarcolemmal C a 2 + - M g 2 + ATPase pump (Carafoli, 1991). Moreover, structural and immunological differences have also been reported to exist between these two pumps (Grover, 1988; Schatzmann, 1989; Grover and Khan , 1992) suggesting that they are, indeed, two distinct isoforms. These inherent differences i n the enzyme activity at the membranes of skeletal muscle fibers may account for the lack of significant staining at the sarcolemma in this study. Results from this study also indicate that skeletal muscle fibers i n the mdx-dystrophic mouse possess a significantly higher level of C a 2 + - M g 2 + pNPPase activity than that observed i n muscle fibers of normal animals. Similar to normal tissue, this activity was partially inhibited i n the mdx muscle fibers by the addit ion of thapsigargin to the incubation medium. The higher enzyme activity observed i n mdx muscle tissue is suggestive of elevated Ca 2 +-sequestering of the SR, however, this higher activity may not be sufficient to reduce sarcoplasmic free C a 2 + levels. It is postulated that a defect i n the SR membrane may not permit efficient sequestering of the C a 2 + and, as a consequence, results i n the leakage of these ions back into the sarcoplasm. This predict ion w o u l d provide an explanation for the elevated sarcoplasmic C a 2 + level reported i n mdx muscle fibers by other investigators (Martonosi, 1967; Duncan, 1978; Turner et al., 1991). It is noteworthy that other investigators have reported a decrease i n functional activity i n SR Ca 2 + -ATPase i n muscle biopsies from patients w i th D M D (Landi et al., 1986; Nagy and Samaha, 1986). From their findings, it was postulated 261 that the increased intracellular free C a 2 + levels may be one of the important factors that contributes to the progressive degeneration of muscle fibers i n this disorder. Al though the mechanism is not completely understood, this influx of C a 2 + may be secondary to a membrane defect (Rowland, 1980). Biochemical studies have shown the increased neutral protease activity affecting the Ca 2 + -ATPase of the SR to be five to ten times higher i n D M D skeletal muscles than i n normal human muscle tissue (Nagy and Samaha, 1986). This increased protease activity caused an observed shift in the SR protein pattern including a decrease i n the 100-kDa band and an increase in the lower-molecular-mass bands wi th 55 and 45 kDa. This fragmentation of the enzyme complex was shown to cause a loss of both ATPase activity and C a 2 + transport associated wi th the SR (Nagy and Samaha, 1986). Contrary to their studies on D M D muscle tissue, the present study revealed a significant increase i n the activity of the SR C a 2 + - M g 2 + pNPPase of mdx muscle tissue. Therefore, it is unlikely that the neutral protease activity is either altered in this condition or affects the C a 2 + -sequestering ability of the SR. Intracellular increases in free C a 2 + concentrations of D M D and mdx skeletal muscle fibers have been thought to be related to defects i n the sarcolemma and/or SR membrane (Martonosi, 1967; Duncan, 1978; Turner et al., 1991). A putative mechanism for this increased concentration has been the open probability of C a 2 + -selective leak channels i n the sarcolemma (Fong et al., 1990). W h e n challenged wi th high C a 2 + , D M D myotubes were the least able to regulate intracellular C a 2 + when compared to normal human and mouse myotubes. C a 2 + leak channel activities of the mdx mouse myotubes, on the other hand, were not as active and showed lower levels of intracellular C a 2 + i n response to a tenfold increase i n extracellular C a 2 + (Fong et al., 1990). These findings may explain, i n part, why muscle necrosis i n D M D is usual ly fatal, whereas the mdx mouse is able to compensate from the initial degenerative insult (Fong et al., 1990). 262 It should be noted i n this study that although the increased activity of C a 2 + -M g 2 + pNPPase activity i n mdx muscle may i n fact reflect the elevated activity of C a 2 + -ATPase, this may not be the actual case since A T P was not used as substrate. While it has been shown by other investigators that A T P and p N P P share a common catalytic site on the SR Ca 2 + -ATPase complex, A T P also interacts w i th a regulatory site indicat ing a complex dependence of the ATPase activity on the substrate (Ribeiro et al., 1980). Differences i n enzyme activity may, therefore, be related to specific requirements of the C a 2 + - p u m p i n g mechanism and not to an overall difference i n phosphorylation rates of these enzymes. Other studies have indicated that the activation of the contractile system by C a 2 + and the maximal force generation were normal i n mdx single-skinned muscle fibers (Takagi et al., 1992). These researchers noted that both C a 2 + uptake of the SR and regulation of the Ca 2 + -induced-Ca 2 + -release mechanism of the SR by C a 2 + were normal. However , contracture by caffeine was reported to be more prominent in mdx than i n control mice indicating increased leakage of C a 2 + into the sarcoplasm. Takagi and coworkers (1992) postulated that this abnormal leakage may also activate the C a 2 + pump of the SR i n the resting state, a process which w o u l d consume extra A T P and disturb energy metabolism. It has also been proposed by another group that intracellular sodium content may be elevated and sodium regulation may be altered or impaired i n mdx skeletal muscle tissue (Dunn et al., 1995). These workers reported an increase i n overall N a + - K + ATPase content; however, their results showed that the elevated pump concentration was unable to compensate entirely for the increased intracellular sodium (Dunn et al., 1995). They suggest that the regulation of ions i n mdx muscles is abnormal and that cell death i n these muscles may be due to abnormal regulation of cell volume. 263 In conclusion, the results from the present study provide evidence that C a 2 + -M g 2 + pNPPase activity i n the SR membranes of extrafusal muscle fibers is distributed i n different regions of the sarcomere and may represent physiologically-diverse calcium-sequestering regions of the SR. Moreover, elevated activity of C a 2 + - M g 2 + pNPPase i n mdx muscle supports the hypothesis that there are functional differences i n the Ca 2 + -sequestering mechanism i n this disease. 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Effect of adenosine triphosphate hydrolysis by lead ion on the histochemical localization of adenosine triphosphatase activity. J. Histochem. Cytochem., 14: 702-710. Mughal , S, A . Cuschieri and A . A . Al-Bader. 1989 Intracellular distribution of C a 2 + -M g 2 + adenosine triphosphatase (ATPase) i n various tissues. J. Anat., 162: 111-124. 268 Nagy, B. and F.J. Samaha 1984a Increased protease activity i n Duchenne dystrophic muscles decreases Ca2+ ATPase i n sarcoplasmic reticulum preparations. A n n . Neurol. , 16: 145. Nagy, B. and F.J. Samaha 1984b Biochemical studies on normal and diseased human muscle sarcoplasmic reticulum: increased protease effects i n muscle diseases. A n n . Neurol. , 16: 401. Nagy, B. and F.J. Samaha 1984c Duchenne dystrophic muscles possess increased protease activity affecting the calcium-adenosine triphosphatase of sarcoplasmic reticulum. IRCS Med . Sci., 12: 828-829. Nagy, B. and F.J. 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Wachstein, M . and E. Meisel 1956 O n the histochemical demonstration of glucose-6-phosphatase. J. Histochem. Cytochem., 4: 592-600. 270 Winegrad, S. 1970 The intracellular site of calcium activation of contraction i n frog skeletal muscle. J. Gen. Physiol., 55: 77-88. Witcome, M . , I. Henderson, A . G . Lee and J.M. East 1992 Mechanism of inhibition of the calcium pump of sarcoplasmic reticulum by thapsigargin. Biochem. J., 283: 525-529. Zinchuk, V.S. and A . V . Bulavka 1992 Ultracytochemical demonstration of C a + + -ATPase activity i n the rat cardiac muscle. Exp. Toxic. Pathol., 44: 150-153. 271 G E N E R A L DISCUSSION The main focus of this investigation was to test the hypothesis that the internal membrane systems in skeletal muscle fibers of three functionally diverse muscles are altered i n mdx-dystrophic mice when compared to their normal age-matched counterparts. This was accomplished by approaches spanning light microscopic , h is tochemical , stereological, t ransmiss ion and high-resolut ion scanning electron microscopic and ultracytochemical methodologies. Significant findings i n Chapter 1 point to alterations i n oxidative capacity in specific muscle fiber types in the E D L , soleus and diaphragm. These results support the hypothesis that mitochondrial oxidative capacities are altered i n skeletal muscles of mdx mice. Quantitative values obtained from the microphotometric analysis of NADH-TR-s ta ined cryosections revealed a differential response of these three muscles to dystrophy. The E D L and soleus muscles of the hindlimb exhibited a decreased oxidative capacity compared to that i n their normal counterparts, whereas an increased oxidative capacity was noted in the muscle fibers of the mdx diaphragm. In both cases, the greatest difference was observed i n the type II fiber populations i n these muscles. It may be argued that measurement of oxidative capacity i n muscle fibers can be variable depending on the type of animal studied (Green et al., 1984) and on the level of physical activity of the animal (Hoppeler, 1986). This was overcome by ensuring that sufficiently h igh numbers of muscle fibers (n=625) and mice (n=5) were examined to reduce the effects of sampling errors and inter-animal variability. Moreover, the application of microphotometric techniques to measure the optical density of histochemical staining also enabled a precise determinat ion of mitochondrial enzyme activity between muscle fibers. 272 Another significant finding i n the first chapter was the smaller cross-sectional areas of fibers i n the mdx diaphragm as compared to the normal diaphragm with the smallest value occuring i n the type Ila fibers. This may represent an adaptive response of the diaphragm to prevent further destruction of its muscle fibers and, thereby, maintain their functional status. O n the other hand, the smaller cross-sectional area of muscle fibers could indicate that the fibers are i n a continual state of turnover which does not allow them to attain a size comparable to that of normal muscle fibers. It w o u l d be of interest to measure the turnover rate of muscle fibers (eg. by autoradiography) in the diaphragm to determine which is the case. Karpati and coworkers (1988) have suggested that the small cross-sectional diameter of a muscle fiber can protect it from the necrotizing effect of the dystrophic gene by offering either mechanical or metabolic advantages to the muscle fiber. If weakening of the sarcolemma is the primary defect in the mdx mouse, a mechanical advantage wou ld be a reduced strain placed on the sarcolemma since the surface-to-volume ratio is much greater than that in large-diameter fibers. The force produced by the lower number of constituent myofibrils w i th in these small muscle fibers wou ld , therefore, be transmitted to a relatively larger sarcolemmal surface area. Another advantage of the small-diameter fibers that w o u l d make them less susceptible to necrosis may be an increased efficiency of uptake and discharge of essential metabolites i n relation to the larger diameter fibers. The elevated oxidative capacity i n the mdx diaphragm observed i n the present study is suggestive of both an elevated metabolic activity and a more efficient transfer of metabolites to and from the fiber. Small-diameter fibers wou ld , therefore, permit a more efficient exchange of oxygen and other metabolites to the mitochondria. Thus, it is predicted that the combination of reduced diameter and increased oxidative capacity i n muscle fibers of the mdx diaphragm represents a structural adaptation that prevents respiratory muscle failure, the underlying cause of death i n human D M D . 273 In Chapter 2, fiber typing of muscle fibers i n the E D L , soleus and diaphragm of normal and mdx mice was undertaken using the ultrastructural parameters of mitochondrial , SR, T-tubule and l i p i d volume density as w e l l as Z line width . Three fiber types were discerned and were classified as either white, intermediate or red. Furthermore, it was found that some of these parameters were more relevant for fiber typing than others and that specific combinations allowed for distinction of fiber types i n these three diverse muscles. Whereas the fast-twitch E D L exhibited the greatest variation i n mitochondrial volume density and Z line widths, the slow-twitch soleus and diaphragm exhibited the greatest differences i n mitochondrial and l i p i d volume density. These parameters, therefore, served as the most reliable distinguishing features of intermediate and red fibers of the slow-twitch soleus and the diaphragm. Al though most of the ultrastructural observations by T E M i n this study fit wel l w i th those of larger animals such as the rat and guinea p ig (Schiaffino et al., 1970; Eisenberg et al., 1976), as a general rule, the mouse skeletal muscle fibers contained higher volume densities of mitochondria and SR elements. These differences i n volume densities suggest that body size may play a role i n determining the extent of organelle development i n muscle fibers. This notion has also been proposed by other investigators w h o per formed comparat ive histochemical and morphological studies on the diaphragm of mammals of varying sizes (Gauthier and Pakykula, 1966; Green et al., 1984). It is important, therefore, to recognize these differences when comparing volume densities of mitochondria, the sarcotubular system and l i p i d as we l l as Z line widths of muscle fibers between species. Ultrastructural features of myofiber degeneration and regeneration in mdx muscle fibers was essentially similar to those reported i n previous investigations of this murine model of dystrophy (Bulfield et a l , 1984; Anderson et al., 1987; Cul len 274 and Jaros, 1988). The earliest pathological alteration in mdx mouse dystrophy was noted to be a dilation of the SR. This is suggestive of an overloading of the SR wi th C a 2 + and may be an attempt by the SR to buffer elevated free C a 2 + i n the sarcoplasm. Whether this is a result of a sarcolemmal disruption or a specific alteration i n the SR membrane, such as failure of C a 2 + channel closing, remains to be elucidated. In v iew of the fact that dystrophin is predicted to protect the sarcolemma from stress induced by muscle contraction and stretch (Petrof et al., 1993; Weller et al., 1990), an intrinsic weakness i n tensile strength of the sarcolemma i n dystrophic muscle fibers has been postulated. This current belief is difficult to prove by ultrastructural methods since small disruptions of the sarcolemma are difficult to detect and, if present, may be a result of artifact due to techniques of specimen preparation. Moreover , that sarcolemmal disrupt ion determines the fate of a muscle fiber may not necessarily be the reason for the init ial insult that results i n necrosis. Therefore, it is essential that further structural and functional investigations be undertaken to determine the subcellular details of dystrophic muscle fibers. The use of H R S E M in Chapter 3 represents the first three-dimensional study of the internal membrane systems in muscle fiber types of the mouse. The goal of this study was to first establish the normal morphological features of these membrane systems and then compare these qualitatively wi th those features of mdx muscle fibers. The A - O - D - O technique, a method by w h i c h non-membrane bounded elements of the sarcoplasm such as myofilaments, cytoskeletal proteins and cytosolic components are extracted, was used i n muscle fiber preparations. The selective preservation of membranous structures was a distinct advantage of this technique and permitted the direct visual izat ion of their fine structure at high resolution. 275 The results of the H R S E M study reveal important spatial relationships of these organelles not normally appreciated in thin sections by T E M . A n important observation by H R S E M was the extremely diversified nature of mitochondrial shape and cristae morphology i n different fiber types and the relationship of mitochondria to the contractile apparatus i n white, intermediate and red muscle fibers. Branching mi tochondr ia l columns and spherical-to-ovoid subsarcolemmal mi tochondr ia observed i n red and intermediate fibers emphasized the fact that morphological variability of these organelles exists wi th in a given muscle fiber. It is predicted that the functional demands of the fiber provide the impetus for mitochondria to become structurally-suited to deliver an effective supply of A T P either to the contractile apparatus and or to energy-dependent processes at the sarcolemma. H R S E M revealed differences i n the disposition of the A band SR i n muscle fibers of the E D L and soleus (i.e. i n fast-twitch vs. slow-twitch fibers). A wel l -developed fenestrated collar wi th tightly anastomosing tubules was always observed i n the three fiber types of the fast-twitch E D L whereas a loose array of SR wi th dilated cisternae was typical of the two kinds of slow-twitch fibers i n the soleus. In addition, the occurence of bare regions of T-tubules i n the red fibers of the soleus are most l ikely indicative of differences in the contractile properties of these two general types of muscle fibers. These spatial arrangements are usually impossible to detect by T E M thin sectioning methods. H R S E M , on the other hand enabled a direct visualization of the course of these tubules wi th in the sarcoplasm by providing the necessary depth of field to elucidate the three dimensionality of these membrane systems. A n understanding of the fine structure of the SR and the composition and function of each region of the SR at the level of the sarcomere is essential to our understanding of the Ca 2 +-transport mechanisms involved i n muscle contraction and relaxation. It is also important to understand how the SR associates wi th other structures i n the muscle fibers such as T-tubules and mitochondria. 276 M d x muscle fibers exhibited similar mitochondrial and sarcotubular system complexity i n the majority of muscle fibers (i.e. i n those that appeared 'healthy') when compared to their normal counterparts. N o obvious pathological defect was detected i n each of the muscle fiber types. Degenerative fibers, on the other hand, showed a disarray of organelles that were often difficult to identify and were presumed dysfunctional. Thus, it was concluded from these observations that the overall distribution and morphology of mitochondria and the sarcotubular system as seen by H R S E M are unaffected i n 'healthy fibers' but not i n degenerative fibers suggesting that these organelles undergo a normal differentiation and maturation process i n muscle fibers of the mdx mouse. The major objective of experiments i n Chapter 4 was to establish a staining procedure that wou ld permit both the ultrastructural localization and measurement of functional activity of the energy dependent Ca 2 + -transporting mechanism of the SR i n skeletal muscle fibers. This method was subsequently used to test the hypothesis that the Ca 2 +-sequestering mechanisms i n mdx muscles are altered as a result of the disease. A significant f inding from this study was that the SR-associated C a 2 + - M g 2 + pNPPase activity in the gastrocnemius was increased by 25% in mdx mice i n comparison to their normal counterparts. These results are indicative of SR alterations i n disease and, therefore, support the hypothesis that the energy-dependent, SR Ca 2 +-sequestering mechanism is altered i n mdx skeletal muscle fibers. There are several possibilities that could account for this observed increase in enzyme activity. First, the cycling of the C a 2 + pump may occur at a higher rate than i n the SR of normal muscle fibers. This may be a result of the expression of functionally different isoforms of either SR C a 2 + ATPase or regulatory proteins such as phospholamban (James et al., 1989) that act on these isoforms. Secondly, a higher density of the enzyme pump per unit membrane of the SR may exist i n mdx muscle fibers. Both of these possibilities may be the result of an adaptation of the SR to a 277 chronic increase i n sarcoplasmic C a 2 + levels i n mdx muscle fibers. It can not be ruled out, however, that alternate enzymes w i t h similar substrate requirements were activated i n other elements of mdx muscle tissue when compared to normal muscle since whole muscle samples were used. This may include the stimulation of other cell types i n the connective tissue interstitium of the whole muscle. It w o u l d be of further interest to apply immunocytochemical staining techniques to more precisely determine the localization and function of SR C a 2 + pump isoforms. Jorgensen and coworkers (1988) have reported that two distinct isoforms of SR C a 2 + ATPase exist i n skeletal muscle, one which predominates i n the slow-twitch type I fibers and another which is present i n fast-twitch fibers. It wou ld therefore be of interest to determine if specific fiber type changes occur i n mdx skeletal muscle fibers by investigating the immunoreactive staining differences of these fast and slow C a 2 + pump isoforms. While the future w i l l foster further exploration into the molecular biology of muscle fibers, it w i l l still require a solid morphological foundation for comparison. Morphological assessment of altered phenotypes by microscopic and stereological methods w i l l serve to reveal the consequences of genetic manipulation and other therapies used to treat diseases such as muscular dystrophy. A cont inuing challenge i n ultrastructural research is to obtain direct visualization of cellular structure and function as they occur i n life. A major pitfall i n most preparative procedures for electron microscopy is the alteration of molecular structure due to the deleterious effects of both chemical fixatives and the high energy of the electron beam. Development of less toxic preparative procedures and more specialized and state-of-the-art instrumentation that are less harmful to cellular and molecular structure w i l l answer pertinent questions about cellular structure and function. Some methods have already been developed to preserve a frozen hydrated state i n tissue including cryo-ultramicrotomy and the use of low-278 temperature specimen stages in the electron microscope. Sjostrom and coworkers (1990) have pioneered the use of these methods to view the myofibrillar ultrastructure of the A band of muscle fibers in greater detail than that previously obtained by conventional plastic-embedding and thin-sectioning methods. Furthermore, the further refinement and utilization of immunochemical and cytochemical methods wil l enable the in situ viewing of the molecular organization of specific subcellular structures. The spatial organization of cellular organelles and their constituent proteins is important in understanding physiological processes. The new era of H R S E M imaging brings the third dimension to cell biology and will continue to provide insights into cellular structure as new preparative procedures are developed. Preserving the antigenicity in tissues for the direct visualization of immunocytochemical probes will give a better understanding of protein structure and function at the macromolecular level. 279 R E F E R E N C E S Anderson, J.E., W . K . Ovalle and B . H . Bressler 1987 Electron microscopic and autoradiographic characterization of hindl imb muscle regeneration i n the mdx mouse. Anat. Rec , 219: 243-257. Bulfield, B. , B .G . Siller, P . A . Wight and K.J . Moore 1984 X chromosome-linked muscular dystrophy (mdx) i n the mouse. Proc. Nat l . Acad . Sci. U S A , 81: 1189-1192. Cul len , M. J . and E. Jaros 1988 Ultrastructure of the skeletal muscle i n the X chromosome-linked dystrophic (mdx) mouse. Compar i son w i t h Duchenne muscular dystrophy. Acta Neuropathol., 77: 69-81. Eisenberg, B.R. and A . M . Kuda 1976 Discrimination between fiber populations i n mammalian skeletal muscle by using ultrastructural parameters. J. Ultrastruct. Res., 54: 76-88. Gauthier, G . and H . A . Padykula 1966 Cytological studies of fiber types i n skeletal muscle. A comparative study of the mammalian diaphragm. J. Ce l l Biol . , 28: 333-354. Green, H.J. , H . Reichmann and D. Pette 1984 Inter- and intraspecies comparisons of fiber type distribution and of succinate dehydrogenase activity i n type I, IIA and LIB fibers of mammalian diaphragms. Histochemistry, 81: 67-73. Hoppeler, H . 1986 Exercise-induced ultrastructural changes in skeletal muscle. Int. J. Sports Med. , 7: 187-204. James, P, M . Inui, M . Tada, M . Chiesi and E. Carafoli 1989 Nature and site of phospholambdan regulation of the C a 2 + of sarcoplasmic reticulum. Nature, 342: 90-92. Jorgensen, A . O . , W. Arno ld , D.R. Pepper, S.D. Kahl , F. Mandel and K.P . Campbell A monoclonal antibody to the Ca 2 + -ATPase of cardiac sarcoplasmic reticulum cross-reacts wi th slow type I but not wi th fast type II canine skeletal muscle fibers: an immunocytochemical and immunochemical study. Ce l l Mot . Cytoskel. , 9: 164-174. Karpati, G . , S. Carpenter and S. Prescott 1988 Small-caliber skeletal muscle fibers do not suffer necrosis i n mdx mouse dystrophy. Muscle Nerve, 11: 795-803. Petrof, B.J., J.B. Shrager, H . H . Stedman, A . M . K e l l y and H . L . Sweeney 1993 Dystrophin protects the sarcolemma from stresses developed dur ing muscle contraction. Proc. Nat l . Acad. Sci. U S A , 90: 3710-3714. Schiaffino, S., V . Hanzl ikova and S. Pierobon 1970 Relations between structure and function i n rat skeletal muscle fibers. J. Cel l B i o l , 47: 107-119. 280 Sjostrdm, M , J . M Squire, P. Luther, E . Morris and A . E d m a n 1990 Cryoultramicrotomy of muscle: improved preservation and resolution of muscle ultrastructure using negatively stained ultrathin cyrosections. J. Microsc, 163: 29-42. Weller, B., G. Karpati and S. Carpenter 1990 Dystrophin-deficient mdx muscle fibers are preferentially vulnerable to necrosis induced by experimental lengthening contractions. J. Neurol. Sci., 100: 9-13. 281 A P P E N D I X 1 Cross-sectional areas (in um 2) and optical density values (in O D units) of N A D H - T R -stained fibers i n the E D L , soleus and diaphragm from each animal (Chapter 1). Values are expressed as the mean of 125 fibers (±standard deviation) per mouse. Area NADH-TR Optical Densi tv Whole Fiber Fiber Core Normal EDL 1 2 3 4 5 1517.4 +599.1 1567.5 ±667.3 1861.5 ±813.3 1812.5 ±778.2 1643.1 ±709.9 0.220 +0.133 0.201 ±0.128 0.199 ±0.143 0.209 ±0.123 0.185 ±0.131 0.149 ±0.098 0.129 ±0.090 0.127 ±0.106 0.134 ±0.086 0.121 ±0.100 1680.4 ±150.8 0.203 ±0.013 0.132 ±0.011 Mdx EDL 1 2 3 4 5 2119.5 ±1285.1 1848.9 ±1064.7 1803.7 ±979.6 1658.4 ±830.2 1916.3 ±1119.9 0.187 ±0.129 0.138 ±0.081 0.154 ±0.101 0.162 ±0.114 0.169 +0.111 0.158 ±0.115 0.108 ±0.068 0.120 ±0.084 0.130 ±0.101 0.140 +0.100 1869.4 +168.8 0.162 ±0.018 0.131 ±0.019 Normal Soleus 1 2 3 4 5 1761.2 ±389.1 2380.7 ±524.0 1901.9 ±437.4 1845.8 ±400.9 2448.9 ±748.4 0.214 ±0.048 0.203 +0.042 0.210 ±0.045 0.243 ±0.058 0.221 ±0.057 0.151 ±0.035 0.132 ±0.030 0.141 ±0.034 0.163 ±0.044 0.157 ±0.048 2067.7 ±321.7 0.218 ±0.015 0.149 ±0.012 Mdx Soleus 1 2 3 4 5 1955.5 ±842.5 2336.6 ±1037.4 1990.6 ±778.4 1693.6 ±837.4 2433.0 ±1094.2 0.192 ±0.033 0.156 ±0.029 0.171 ±0.033 0.157 ±0.029 0.191 ±0.034 0.146 ±0.032 0.124 ±0.029 0.133 ±0.033 0.121 ±0.028 0.147 ±0.030 2081.9 ±301.3 0.173 ±0.018 0.134 ±0.012 Normal Diaphragm 1 2 3 4 5 1004.0 ±455.8 1181.6 ±575.2 1160.8 ±455.2 961.4 ±339.6 970.4 ±401.8 0.224 ±0.054 0.234 ±0.064 0.225 ±0.053 0.227 ±0.046 0.230 ±0.060 0.172 ±0.052 0.190 ±0.068 0.175 ±0.056 0.169 ±0.041 0.184 ±0.065 1055.6 ±106.9 0.228 ±0.004 0.178 ±0.009 Mdx Diaphragm 1 2 3 4 5 794.6 ±453.6 714.1 ±334.3 718.6 ±360.3 795.5 ±419.8 908.7 ±461.2 0.257 ±0.070 0.246 ±0.052 0.249 ±0.060 0.246 ±0.063 0.224 ±0.065 0.233 ±0.078 0.211 ±0.059 0.207 ±0.058 0.213 ±0.069 0.191 ±0.073 786.3 +78.9 0.244 ±0.012 0.211 ±0.015 282 A P P E N D I X 2 Mean cross-sectional areas (in um 2) and optical density values (in O D units) of type I, type Ila and type l i b fibers i n the E D L from each animal stained for N A D H - T R (Chapter 1). Standard deviations from the average are indicated. NADH-TR Optical Density n Area Whole Fiber Fiber Core Normal EDL 1. Type I Type Ila Type lib 4 66 55 750.7 ±141.8 1982.2 ±302.9 1015.4 ±385.3 0.406 ±0.044 0.112 ±0.048 0.335 ±0.086 0.309 ±0.016 0.073 ±0.028 0.228 ±0.078 2. Type 1 Type Ila Type lib 2 72 51 659.3 ±255.2 1991.0 ±494.9 1005.4 ±376.3 0.404 ±0.065 0.106 ±0.042 0.327 ±0.085 0.284 ±0.075 0.067 ±0.025 0.212 ±0.074 3. Type I Type Ila Type lib 1 70 54 626.2 2449.3 ±512.4 1122.5 ±405.0 0.416 0.092 ±0.057 0.334 ±0.092 0.310 0.053 ±0.038 0.220 ±0.088 4. Type I Type Ila Type lib 2 76 47 526.6 ±157.2 2290.8 ±563.2 1093.6 ±369.9 0.328 ±0.127 0.123 ±0.048 0.341 ±0.079 0.248 ±0.050 0.079 ±0.029 0.221 ±0.072 5 Type I Type Ila Type lib 1 75 49 598.4 2097.0 ±476.0 969.8 ±380.9 0.465 0.092 ±0.042 0.321 ±0.087 0.344 0.055 ±0.027 0.218 ±0.086 M d x EDL 1 Type I Type Ila Type lib 1 57 67 753.3 2704.0 ±1285.9 1642.6 ±1070.3 0.301 0.081 ±0.036 0.276 ±0.111 0.217 0.064 ±0.027 0.236 ±0.102 2. Type I Type Ila Type lib 2 80 43 562.3 ±47.5 2137.5 ±1126.8 1371.8 ±698.6 0.285 ±0.029 0.092 ±0.046 0.215 ±0.063 0.244 ±0.013 0.071 ±0.037 0.171 ±0.059 3. Type I Type Ila Type lib 1 57 40 527.4 2404.8 ±977.9 1311.4 ±639.4 0.314 0.071 ±0.034 0.221 ±0.084 0.276 0.056 ±0.025 0.173 ±0.077 4. Type I Type Ila Type lib 1 67 57 750.6 2007.2 ±850.6 1264.3 ±595.9 0.380 0.078 ±0.044 0.258 ±0.089 0.361 0.059 ±0.035 0.210 ±0.089 5. Type 1 Type Ila Type lib 2 56 67 292.5 ±57.7 2452.7 ±1164.2 1516.3 ±860.5 0.275 ±0.071 0.080 ±0.033 0.240 ±0.099 0.259 ±0.004 0.062 ±0.026 0.201 ±0.093 283 APPENDIX 3 Mean cross-sectional areas (in um2) and optical density values (in O D units) of type I, type Ila and type lib fibers in the soleus from each animal stained for N A D H - T R (Chapter 1). Standard deviations from the average are indicated. n Area NADH-TR Optical Densitv Whole Fiber Fiber Core Normal Soleus 1 Type I Type Ila Type lib 33 82 10 1976.3 ±509.8 1699.9 ±268.9 1555.1 ±509.6 0.160 0.234 0.228 ±0.032 ±0.037 ±0.042 0.117 0.162 0.167 ±0.028 ±0.030 ±0.028 2 Type I Type Ila Type lib 46 68 11 2546.3 ±485.2 2197.7 ±442.7 2819.2 ±683.8 0.162 0.225 0.230 ±0.021 ±0.031 ±0.045 0.106 0.149 0.137 ±0.019 ±0.022 ±0.032 3 Type I Type Ila Type lib 47 75 3 1815.0 1927.0 2636.2 ±334.3 ±472.5 ±149.8 0.166 0.235 0.272 ±0.026 ±0.030 ±0.035 0.111 0.158 0.176 ±0.026 ±0.025 ±0.009 4 Type I Type Ila Type lib 42 70 13 2001.6 1721.3 2012.7 ±349.1 ±358.0 ±549.1 0.180 0.276 0.266 ±0.034 ±0.040 ±0.036 0.118 0.184 0.190 ±0.029 ±0.030 ±0.033 5 Type I Type Ila Type lib 40 55 30 2456.5 2209.4 2877.8 ±687.2 ±531.0 ±967.4 0.169 0.252 0.232 ±0.028 ±0.042 ±0.062 0.124 0.178 0.162 ±0.021 +0.044 ±0.059 Mdx Soleus 1 Type 1 Type Ila Type lib 38 67 20 2022.8 2054.9 1494.3 ±793.5 ±794.5 ±970.8 0.169 0.205 0.194 ±0.026 ±0.028 ±0.041 0.130 0.155 0.150 ±0.028 ±0.028 ±0.041 2 Type I Type Ila Type lib 54 54 17 2308.1 2485.8 1953.5 ±930.5 ±1190.7 ±736.8 0.143 0.167 0.163 ±0.027 ±0.028 ±0.021 0.116 0.132 0.129 ±0.029 ±0.027 ±0.026 3 Type I Type Ila Type lib 45 55 25 1732.9 2281.6 1814.0 ±770.2 ±703.1 ±761.3 0.152 0.182 0.179 ±0.032 ±0.028 ±0.031 0.118 0.142 0.140 ±0.036 ±0.028 ±0.030 4 Type I Type Ila Type lib 38 61 26 1555.7 1881.7 1453.9 ±676.9 ±837.7 ±969.8 0.136 0.169 0.166 ±0.021 ±0.032 ±0.032 0.103 0.127 0.132 ±0.023 ±0.026 ±0.027 5 Type I Type Ila Type lib 56 45 24 2581.6 2518.1 1926.4 ±963.5 ±1196.2 ±1077.9 0.173 0.211 0.197 ±0.028 ±0.028 ±0.035 0.132 0.162 0.155 ±0.025 ±0.028 ±0.029 284 A P P E N D I X 4 Mean cross-sectional areas (in um 2) and optical density values (in O D units) of type I, type Ila and type l ib fibers i n the diaphragm from each animal stained for N A D H - T R (Chapter 1). Standard deviations from the average are indicated. Normal Diaphragm 1 Type I Type Ila Type lib 2. 3. 4. 5. Type I Type Ila Type lib Type I Type Ila Type lib Type I Type Ila Type lib Type I Type Ila Type lib Mdx Diaphragm 1. 5. Type I Type Ila Type lib Type I Type Ila Type lib Type I Type Ila Type lib Type I Type Ila Type lib Type I Type Ila Type lib n 11 75 39 9 74 42 11 66 •48 9 70 46 16 63 46 26 58 41 24 67 34 20 53 52 20 39 66 25 36 64 Area 690.3 +218.6 1168.2 ±500.9 776.8 ±205.1 686.2 ±92.8 1463.1 ±557.2 791.8 ±305.9 870.4 ±317.9 1394.9 ±474.9 905.4 ±223.6 864.0 ±315.3 1093.1 ±366.7 779.9 ±177.4 741.4 ±231.4 1161.5 ±436.8 788.2 ±245.8 873.3 ±467.1 702.8 ±324.0 874.5 ±573.6 726.4 ±465.5 714.9 +305.3 703.6 ±286.6 784.5 ±389.3 729.0 ±407.4 682.6 ±294.8 755.0 ±547.1 790.9 ±414.8 810.5 ±384.0 1133.4 ±538.1 895.5 ±411.5 828.3 ±433.1 NADH-TR Optical Density Whole Fiber Fiber Core 0.221 ±0.025 0.210 ±0.049 0.250 ±0.058 0.247 ±0.043 0.207 ±0.059 0.278 ±0.049 0.245 ±0.041 0.199 ±0.046 0.256 ±0.046 0.249 ±0.038 0.208 ±0.042 0.251 ±0.041 0.265 ±0.060 0.214 ±0.052 0.239 ±0.064 0.262 ±0.086 0.252 ±0.058 0.261 ±0.074 0.237 ±0.043 0.247 ±0.049 0.250 ±0.063 0.252 ±0.035 0.237 ±0.067 0.259 ±0.058 0.247 ±0.057 0.264 ±0.062 0.235 ±0.064 0.224 ±0.053 0.217 ±0.069 0.228 ±0.068 0.181 ±0.023 0.155 ±0.045 0.203 ±0.057 0.229 ±0.046 0.157 ±0.059 0.241 ±0.047 0.198 ±0.040 0.146 ±0.044 0.210 +0.051 0.184 ±0.029 0.150 ±0.037 0.193 ±0.035 0.235 ±0.074 0.160 ±0.052 0.199 ±0.063 0.255 ±0.084 0.218 ±0.072 0.240 ±0.081 0.205 ±0.062 0.210 ±0.052 0.217 ±0.070 0.206 ±0.050 0.199 ±0.065 0.216 ±0.052 0.222 ±0.071 0.230 ±0.068 0.199 +0.068 0.196 ±0.064 0.179 ±0.074 0.195 ±0.076 285 A P P E N D I X 5 Raw T E M point count data of mitochondria and SR-T system (percentage of fiber core i n longitudinal sections) and Z line measurements (in nm) for muscle fibers of the normal E D L (Chapter 2). Normal EDL Mouse Mitochondria SR T-tubule SR-T Lipid Z-line 1 2.49 9.67 0.73 10.40 0 71.20 2.56 9.74 0.51 10.25 0 71.20 4.91 7.84 0.73 8.57 0 71.20 5.13 7.55 . 0.51 8.06 0 74.80 9.42 7.20 0.58 7.78 0 106.84 10.29 7.79 0.43 8.24 0 96.16 2 2.86 6.92 0.68 7.60 0 64.10 2.93 9.38 0.59 9.97 0 64.10 5.40 9.26 0.57 9.83 0 89.04 6.02 8.90 0.36 9.26 0 92.60 9.34 7.73 0.44 8.17 0 103.28 11.54 7.45 0.53 7.98 0 96.16 3 3.36 8.65 0.51 9.16 0 71.20 3.81 10.70 0.73 11.43 0 64.10 7.62 8.94 0.44 9.38 0 78.35 9.05 8.32 0.39 8.71 0 80.25 10.32 7.88 0.45 8.33 0 103.28 13.60 8.64 0.59 9.23 0 97.94 4 2.64 9.74 0.37 10.11 0 64.10 2.71 9.44 0.48 9.92 0 64.10 6.44 8.23 0.57 8.80 0 71.20 7.04 8.31 0.42 8.73 0 71.20 10.79 7.54 0.49 8.03 0 97.94 12.22 7.21 0.35 7.56 0 103.28 5 2.56 8.67 0.44 9.11 0 64.10 3.02 9.33 0.59 9.92 0 64.10 5.21 8.45 0.53 8.98 0 71.20 6.89 7.45 0.42 7.87 0 71.20 9.90 7.34 0.36 7.70 0 106.84 11.31 6.88 0.42 7.30 0 97.94 286 A P P E N D I X 6 Raw T E M point count data of mitochondria and SR-T system (percentage of fiber core i n longitudinal sections) and Z line measurements (in nm) for muscle fibers of the normal soleus (Chapter 2). Normal Soleus Mouse Mitochondria SR T-tubule SR-T Lipid Z-line 1 8.69 6.91 0.36 7.27 0 110.40 9.47 5.55 0.57 6.12 0 2 106.85 9.69 7.34 0.43 7.77 0 28 101.50 12.61 5.70 0.57 6.27 0 57 106.85 13.60 5.63 0.50 6.13 0 36 103.28 2 9.19 5.56 0.50 6.06 0 106.85 10.04 6.34 0.36 6.70 0 71 103.28 12.61 4.49 0.43 4.92 0 29 106.85 12.94 5.06 0.43 5.49 0 53 106.85 16.03 4.49 0.29 4.78 0 68 103.28 3 6.27 4.42 0.36 4.78 0 28 110.76 7.26 4.91 0.36 5.27 0 57 110.41 8.26 4.91 0.50 5.41 0 50 113.97 8.62 6.77 0.36 7.13 0 14 97.94 10.47 3.49 0.21 3.70 0 71 113.97 4 9.43 6.27 0.50 7.27 0 15 106.85 9.76 5.98 0.64 6.62 0 28 106.85 10.97 4.99 0.28 5.27 0 71 106.85 11.18 5.48 0.50 5.98 0 43 99.72 12.96 6.77 0.36 7.13 0 64 106.85 5 6.67 4.91 0.66 5.57 0 106.85 8.19 5.77 0.57 6.34 0 106.85 9.12 6.20 0.50 6.70 0 28 97.94 9.90 6.77 0.36 7.13 0 50 106.85 10.11 6.34 0.64 6.97 0 36 106.85 287 A P P E N D I X 7 Raw T E M point count data of mitochondria and SR-T system (percentage of fiber core i n longitudinal sections) and Z line measurements (in nm) for muscle fibers of the normal diaphragm (Chapter 2). Normal Diaphragm Mouse Mitochondria SR T-tubule SR-T Lipid Z-lirte 1 6.74 9.23 0.66 9.89 r 0 71.23 7.69 7.33 0.73 8.06 0.11 71.23 8.57 7.69 0.73 8.42 0 71.23 9.08 7.62 0.59 8.21 0.14 74.80 11.43 9.16 0.59 9.75 0.11 71.23 2 8.82 6.41 0.62 7.03 0.28 71.23 9.92 7.69 0.88 8.57 0 74.80 12.43 8.35 1.10 9.45 0.28 71.23 12.92 8.57 0.88 9.45 0.21 74.80 13.61 9.30 0.59 9.89 0.28 78.35 3 6.22 8.24 0.59 8.83 0.11 71.23 8.23 7.53 0.88 8.41 0.21 74.79 11.52 9.41 0.56 9.97 0.28 80.22 12.54 7.88 0.45 8.33 0.28 74.79 12.98 7.25 0.73 7.98 0.11 74.80 4 5.89 8.42 0.41 8.83 0 71.23 7.93 8.42 0.62 9.04 0.21 74.80 8.59 7.35 0.52 7.87 0.11 71.23 10.53 8.34 0.69 9.01 0.28 74.80 11.63 6.25 0.52 6.77 0.21 80.23 5 6.32 9.45 0.66 10.11 0.11 73.42 8.51 8.42 0.59 9.01 0.11 75.22 9.56 7.89 0.42 8.31 0.21 80.23 14.32 8.21 0.52 8.73 0.35 84.23 16.31 7.12 0.41 7.53 0.28 84.23 288 A P P E N D I X 8 Raw T E M point count data of mitochondria and SR-T system (percentage of fiber core i n longitudinal sections) and Z line measurements (in nm) for muscle fibers of the mdx E D L (Chapter 2). Mdx EDL Mouse Mitochondria SR T-tubule SR-T Lipid Z-lfne 1 2.86 9.52 0.81 10.33 17 • 1 0 40.00 2.27 9.60 0.44 10.04 0 42.74 3.59 10.04 0.59 10.63 0 42.74 5.67 7.06 0.52 7.58 0 71.23 8.42 8.24 0.41 8.65 0 78.35 2 2.12 10.26 0.59 10.85 0 40.00 2.33 10.99 0.73 11.72 0 42.74 3.21 10.11 0.73 10.84 0 71.23 4.98 7.18 0.59 7.77 0 71.23 8.55 8.13 0.37 8.50 0 78.35 3 5.27 7.18 0.51 7.69 0 42.74 5.79 11.06 0.66 11.72 0 71.23 6.59 7.91 0.44 8.35 0 71.23 7.47 10.92 0.81 11.73 0 64.10 9.38 8.50 0.59 9.09 0 74.79 4 3.75 7.41 0.59 8..00 0 42.74 4.20 8.50 0.51 9.01 0 42.74 4.80 8.25 0.37 8.62 0 64.10 5.29 7.11 0.73 7.84 0 71.23 7.36 8.50 0.44 8.94 0 81.92 5 2.45 8.41 0.62 9.03 0 40.00 3.25 7.35 0.37 7.82 0 42.74 6.45 9.24 0.42 9.66 0 64.10 7.74 7.92 0.42 8.34 0 71.23 9.44 7.25 0.57 7.82 0 74.79 289 A P P E N D I X 9 Raw T E M point count data of mitochondria and SR-T system (percentage of fiber core in longitudinal sections) and Z line measurements (in nm) for muscle fibers of the mdx soleus (Chapter 2). Mdx Soleus Mouse Mitochondria SR T-tubule SR-T Lipid Z-line 1 4.84 4.62 0.44 5.06 0 07 106.85 5.13 6.29 0.51 6.80 0 07 106.85 5.20 5.42 0.51 5.93 0 22 106.85 5.47 3.96 0.59 4.55 0 07 113.97 6.82 5.24 0.44 5.68 0 11 106.85 2 4.92 5.13 0.37 5.50 0 101.50 5.79 3.96 0.51 4.47 0 07 101.50 6.30 4.40 0.51 4.91 0 07 106.85 6.45 4.88 0.37 5.25 0 15 104.22 6.74 4.91 0.28 5.19 0 07 113.97 3 5.86 5.41 0.43 5.84 0 07 110.41 7.79 4.06 0.36 4.42 0 110.41 7.90 5.12 0.39 5.51 0 11 106.85 8.13 4.70 0.50 5.20 0 22 106.85 8.50 4.24 0.36 4.60 0 22 106.85 4 5.83 4.87 0.50 5.37 0 11 104.22 6.14 4.76 0.37 5.13 0 15 106.85 6.92 4.82 0.43 5.25 0 07 106.85 7.84 3.92 0.28 4.20 0 07 110.41 10.70 4.49 0.21 4.70 0 07 106.85 5 4.91 3.74 0.37 4.11 0 07 101.50 6.89 4.76 0.51 5.27 0 22 106.85 7.42 5.21 0.62 5.83 0 11 106.85 8.01 6.24 0.42 6.66 0 07 106.85 9.44 5.26 0.51 5.77 0 22 106.85 290 A P P E N D I X 10 Raw T E M point count data of mitochondria and SR-T system (percentage of fiber core in longitudinal sections) and Z line measurements (in nm) for muscle fibers of the mdx diaphragm (Chapter 2). Mdx Diaphragm Mouse Mitochondria SR T-tubule SR-T Lipid Z-line 1 5.79 8.21 0.21 8.42 0.07 71.23 6.14 8.14 0.29 8.43 0 71.23 6.22 6.71 0.57 7.28 0.12 71.23 8.07 7.07 0.57 7.64 0 71.23 15.00 5.36 0.43 5.79 0.46 81.92 2 5.11 5.00 0.43 5.43 0.21 71.23 7.42 6.36 0.43 6.79 0.07 71.23 9.36 8.57 0.71 9.28 0.14 74.25 10.12 6.24 0.61 6.65 0.21 85.83 14.31 5.82 0.58 6.40 0.28 85.83 3 6.19 6.45 0.44 6.89 0.07 71.23 8.12 7.62 0.50 8.12 0.22 71.23 10.07 8.64 0.43 9.07 0.71 74.25 13.57 7.62 0.43 8.05 0.61 89.04 16.50 6.64 0.36 7.00 0.54 91.25 4 6.32 7.29 0.43 7.72 0.12 64.11 7.13 6.71 0.21 6.92 0.07 71.23 11.65 7.57 0.57 8.14 0.22 64.11 13.63 9.07 0.64 9.71 0.59 78.35 14.45 6.34 0.28 6.62 0.83 78.35 5 5.07 7.57 0.36 7.93 0 64.11 5.36 6.79 0.57 7.36 0 72.13 7.81 6.89 0.42 7.31 0.11 74.22 9.69 5.20 0.21 5.41 0.36 78.98 13.72 6.21 0.28 6.49 0.64 81.91 291 

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