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Alterations in fast and slow-twitch muscles of genetically dystrophic mice with special reference to.. 1987

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ALTERATIONS IN FAST AND SLOW-TWITCH MUSCLES OF GENETICALLY DYSTROPHIC MICE WITH SPECIAL REFERENCE TO PARVALBUMIN By MARJORIE ISABELLE JOHNSON B.Sc, The University of Waterloo, 1978. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF GRADUATE STUDIES (Department of Anatomy) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1987 © Marjorie Isabelle Johnson, 1987. In p resen t i ng this thesis in part ial fu l f i lment o f the requ i remen ts for an a d v a n c e d d e g r e e at t he Un ivers i ty o f Br i t ish C o l u m b i a , I agree that t he Library shal l m a k e it f reely avai lable for re fe rence and s tudy . I fur ther agree that pe rm iss ion for ex tens i ve c o p y i n g o f th is thesis for scho la r l y p u r p o s e s may be g ran ted by the h e a d o f m y depa r tmen t o r by his o r her representa t i ves . It is u n d e r s t o o d that c o p y i n g o r pub l i ca t i on o f this thesis fo r f inanc ia l ga in shal l not b e a l l o w e d w i t h o u t m y wr i t t en p e r m i s s i o n . T h e Un ivers i ty o f Bri t ish C o l u m b i a 1956 M a i n M a l l V a n c o u v e r , C a n a d a D e p a r t m e n t V 6 T 1Y3 DE-6(3/81) A B S T R A C T M u s c u l a r d y s t r o p h y is a gene t i c d isease w h i c h a f f e c t s the m o r p h o l o g y , p h y s i o l o g y a n d b i o c h e m i c a l na tu re o f the m u s c l e f i b e r . T h i s s t u d y was d e s i g n e d to e x a m i n e the p r o g r e s s i v e e f f e c t s o f m u s c u l a r d y s t r o p h y o n the d i f f e r e n t i a t i o n p rocess o f ske le ta l m u s c l e . C h a p t e r 1 e x a m i n e s the n e o n a t a l d e v e l o p m e n t o f m u s c l e s p i n d l e s a n d t he i r i n t r a f u s a l f i b e r s i n the so leus a n d e x t e n s o r d i g i t o r u m l ongus ( E D L ) o f g e n e t i c a l l y d y s t r o p h i c m i c e a c c o r d i n g to h i s t o c h e m i c a l , q u a n t i t a t i v e , a n d u l t r a s t r u c t u r a l p a r a m e t e r s . D e s p i t e a l t e ra t i ons i n the s u r r o u n d i n g e x t r a f u s a l f i b e r s , m u s c l e s p i n d l e s a n d t h e i r i n t r a f u s a l f i b e r s a p p e a r e d e n z y m a t i c a l l y a n d h i s t o l o g i c a l l y u n a f f e c t e d i n i n c i p i e n t stages o f m u r i n e d y s t r o p h y . I n the s e c o n d c h a p t e r the d i s t r i b u t i o n a n d c o n c e n t r a t i o n o f p a r v a l b u m i n ( P V ) , a c a l c i u m - b i n d i n g p r o t e i n , i n 32 a n d 2 - w e e k - o l d d y s t r o p h i c m i c e was m a p p e d b y i m m u n o h i s t o c h e m i c a l a n d b i o c h e m i c a l p r o c e d u r e s . T h e n u m b e r o f p a r v a l b u m i n - i m m u n o r e a c t i v e f i b e r s was s i g n i f i c a n t l y r e d u c e d i n the a d u l t d y s t r o p h i c E D L bu t s l i g h t l y i n c r e a s e d i n the a d u l t d y s t r o p h i c so leus . N o d i f f e r e n c e s b e t w e e n s t ra ins w e r e o b s e r v e d i n the 2 - w e e k s a m p l e s . T h e s e f i n d i n g s w e r e s u p p o r t e d b y r o u t i n e m y o s i n A T P a s e h i s t o c h e m i s t r y . P a r v a l b u m i n was i so l a t ed o n S D S - P A G E gels a n d the c o n c e n t r a t i o n o f P V was e s t i m a t e d b y a R I A . T h e s e resu l ts c o n f i r m e d the i m m u n o h i s t o c h e m i c a l da ta i n that P V c o n t e n t was d r a m a t i c a l l y r e d u c e d i n the a d u l t d y s t r o p h i c E D L a n d s i g n i f i c a n t l y i n c r e a s e d i n the d y s t r o p h i c so leus . N o changes w e r e d e t e c t e d i n the samp les o f the 2 - w e e k - o l d m u s c l e s . T h e s i m i l a r i t y i n the d i s t r i b u t i o n a n d c o n t e n t o f P V b e t w e e n the fas t a n d s l o w d y s t r o p h i c m u s c l e s at 32 w e e k s o f age suggests a n a l t e r a t i o n i n the d i s t r i b u t i o n a n d p h e n o t y p i c e x p r e s s i o n o f f i b e r t ypes i n m u s c u l a r d y s t r o p h y a n d s u p p o r t s the h y p o t h e s i s that d y s t r o p h y a l ters the n o r m a l d i f f e r e n t i a t i o n p rocess o f s ke le ta l m u s c l e . i i T A B L E O F C O N T E N T S Page A b s t r a c t i i T a b l e o f C o n t e n t s i i i L i s t o f T a b l e s v i L i s t o f F i g u r e s v i i A c k n o w l e d g e m e n t x D e d i c a t i o n x i G e n e r a l I n t r o d u c t i o n 1 A n i m a l M o d e l s 1 i ) H i s t o l o g i c a l F e a t u r e s 2 i i ) F i b e r T y p e s 4 i i i ) P h y s i o l o g i c a l a n d B i o c h e m i c a l C h a n g e s 6 S ta temen t o f the O b j e c t i v e s 9 Chapter 1 P o s t n a t a l d e v e l o p m e n t o f m u s c l e s p i n d l e s i n g e n e t i c a l l y d y s t r o p h i c m i c e . I n t r o d u c t i o n 10 M a t e r i a l a n d M e t h o d s 12 H i s t o c h e m i s t r y 12 Q u a n t i t a t i o n 13 E l e c t r o n M i c r o s c o p y 13 O b s e r v a t i o n s 15 H i s t o c h e m i s t r y 15 M o r p h o l o g y a n d u l t r a s t r u c t u r e 18 Q u a n t i t a t i o n a n d d i s t r i b u t i o n 25 D i s c u s s i o n 35 i i i Spindles in slow-twitch versus fast-twitch muscles Morphogenesis Functional and clinical considerations 38 40 41 Chapter 2 Biochemical and Immunochemical localization of parvalbumin Introduction 44 Historical Background of Parvalbumin 44 Evolution 45 Biochemistry of Parvalbumin 45 Calcium and Magnesium Binding 47 Functional Aspects 48 Intracellular Distribution 53 Quantitation of Parvalbumin 55 Material and Methods 58 General 58 Characterization of Parvalbumin 59 i) Tissue Preparation 62 ii) Two-Dimensional Electrophoresis 62 Antisera Production 64 Charaterization of the Antibody 65 i) Enzyme-Linked Immunosorbant Assay (ELISA) 65 ii) Tissue Preparation for Immunochemical Studies 66 iii) Immunoblots 66 iv) Radioimmunoassay 70 Specific Methods 74 Immunohistochemistry 74 Histochemistry 75 Statistical Analysis 76 iv D e t e r m i n a t i o n o f P a r v a l b u m i n C o n t e n t 77 i ) P r o t e i n D e t e r m i n a t i o n 77 i i ) R a d i o i m m u n o a s s a y 77 R e s u l t s 84 C h a r a c t e r i z a t i o n o f P a r v a l b u m i n 84 C h a r a c t e r i z a t i o n o f A n t i b o d y 93 I m m u n o h i s t o c h e m i s t r y 100 i ) A d u l t E D L 107 i i ) A d u l t So leus , 113 i i i ) T w o w e e k E D L 118 i v ) T w o w e e k so leus 119 S u m m a r y o f I m m u n o h i s t o c h e m i c a l R e s u l t s 124 P a r v a l b u m i n C o n t e n t 132 D e v e l o p m e n t a l C h a n g e s 133 D i s c u s s i o n 145 I m m u n o h i s t o c h e m i s t r y 145 i ) M u s c l e S p i n d l e s 148 P a r v a l b u m i n C o n t e n t 150 G e n e r a l D i s c u s s i o n 157 B i b l i o g r a p h y 165 v LIST OF T A B L E S I. Comparison of mean spindle number and mean spindle index for normal (N) and dystrophic (DY) soleus and E D L muscles at 1, 2, and 3 weeks of age. II. Amino acid composition of parvalbumin in the rat and mouse. III. Summary of parvalbumin content in various species and tissues. IV. Summary of fixation and sectioning methods investigated for the immunohistochemical localization of parvalbumin. V. Distribution of fiber types according to parvalbumin immunoreactivity for normal (N) and dystrophic (DY) muscles at 32 and 2 weeks of age. VI. Distribution of fiber types according to myosin ATPase reactivity for normal (N) and dystrophic (DY) muscles at 32 and 2 weeks of age. VII. Correlation of fiber classifications for parvalbumin and myosin ATPase staining procedures. VIII Mean values for parvalbumin content in 32 week normal (N) and dystrophic (DY) muscle samples. IX. Mean values for parvalbumin content in 2 week normal (N) and dystrophic (DY) muscle samples. vi L I S T O F F I G U R E S Page 1-2 . M y o s i n A T P a s e o f i n t r a f u s a l f i b e r s i n n o r m a l so leus a n d E D L m u s c l e s at 1 w e e k o f age 17 3 - 4 . M u s c l e s p i n d l e i n n o r m a l 3 w e e k o l d s o l e u s , m y o s i n A T P a s e a c i d a n d a l k a l i n e p r e i n c u b a t i o n 20 5 - 6 . M u s c l e s p i n d l e i n d y s t r o p h i c so leus at 3 w e e k s o f age , m y o s i n A T P a s e a c i d a n d a l k a l i n e p r e i n c u b a t i o n 20 7 - 8 . M u s c l e s p i n d l e i n a n o r m a l E D L at 3 w e e k s o f age , m y o s i n A T P a s e a c i d a n d a l k a l i n e p r e i n c u b a t i o n 22 9 - 1 0 M u s c l e s p i n d l e i n a d y s t r o p h i c E D L at 3 w e e k s o f age , m y o s i n A T P a s e a c i d a n d a l k a l i n e p r e i n c u b a t i o n 22 11. S c h e m a t i c d i a g r a m o f i n t r a f u s a l f i b e r s t a i n i n g p a t t e r n 24 12. L i g h t m i c r o g r a p h o f 3 - w e e k d y s t r o p h i c so leus 27 13. E l e c t r o n m i c r o g r a p h o f a j u x t a e q u a t o r i a l s p i n d l e f r o m a 3 - w e e k d y s t r o p h i c so leus 29 14. E l e c t r o n m i c r o g r a p h o f an e q u a t o r i a l s p i n d l e f r o m a 3 - w e e k d y s t r o p h i c E D L 29 15. L o n g i t u d i n a l a n d t ransverse r e c o n s t r u c t i o n o f s p i n d l e d i s t r i b u t i o n i n n o r m a l so leus a n d E D L musc les 33 16. L o n g i t u d i n a l a n d t ransve rse r e c o n s t r u c t i o n o f s p i n d l e d i s t r i b u t i o n i n d y s t r o p h i c so leus a n d E D L musc les 35 17. S c h e m a t i c d i a g r a m i l l u s t r a t i n g the p r o p o s e d f u n c t i o n o f p a r v a l b u m i n 51 18. G e n e r a l m e t h o d o l o g y o u t l i n e 61 19. E n z y m e - l i n k e d i m m u n o a b s o r b a n t test c u r v e s 68 20 . S c h e m a t i c o u t l i n e o f the r a d i o i m m u n o a s s a y p r o c e d u r e 72 v i i 2 1 . S t a n d a r d c u r v e f o r p r o t e i n assay o f a d u l t m u s c l e samp les 79 2 2 . S t a n d a r d c u r v e f o r p r o t e i n assay o f n e o n a t a l m u s c l e samp les 81 2 3 . C o m i g r a t i o n o f p a r v a l b u m i n w i t h E D L samp les 86 24 . C o m i g r a t i o n o f p a r v a l b u m i n w i t h so leus s a m p l e s 88 2 5 . I soe lec t r i c f o c u s i n g gels o f n o r m a l a n d d y s t r o p h i c a d u l t s a m p l e s 90 26 . I soe lec t r i c f o c u s i n g gels o f n o r m a l a n d d y s t r o p h i c 2 - w e e k s a m p l e s 92 2 7 . T w o - d i m e n s i o n a l P A G E o f 2 - w e e k n o r m a l E D L 95 28 . T w o - d i m e n s i o n a l P A G E o f 2 - w e e k d y s t r o p h i c E D L 95 2 9 . T w o - d i m e n s i o n a l P A G E o f 2 - w e e k n o r m a l so leus 97 30 . T w o - d i m e n s i o n a l P A G E o f 2 - w e e k d y s t r o p h i c so leus 97 3 1 . O n e - d i m e n s i o n a l P A G E used f o r i m m u n o b l o t t i n g 99 3 2 . I m m u n o b l o t o f 32 w e e k o l d m u s c l e samp les 99 33 . S t a n d a r d c u r v e f o r R I A o f c r o s s - r e a c t i v i t y 102 34. E n l a r g e m e n t o f o n e - d i m e n s i o n a l P A G E s h o w n i n F i g u r e 50 104 3 5 . O n e - d i m e n s i o n a l P A G E a n d i m m u n o b l o t o f 2 - w e e k - o l d m u s c l e samp les 106 36. I m m u n o h i s t o c h e m i s t r y o f p a r v a l b u m i n i n n o r m a l 3 2 - w e e k - o l d E D L 109 37 . P a r v a l b u m i n d i s t r i b u t i o n i n d y s t r o p h i c 3 2 - w e e k - o l d E D L 109 38 . M y o s i n A T P a s e o f n o r m a l 3 2 - w e e k E D L 112 39 . M y o s i n A T P a s e o f d y s t r o p h i c 3 2 - w e e k E D L 112 40 . P a r v a l b u m i n l o c a l i z a t i o n i n n o r m a l 3 2 - w e e k so leus 115 4 1 . P a r v a l b u m i n l o c a l i z a t i o n i n d y s t r o p h i c 3 2 - w e e k so leus 115 4 2 . M y o s i n A T P a s e o f n o r m a l 3 2 - w e e k so leus 117 4 3 . M y o s i n A T P a s e o f d y s t r o p h i c 3 2 - w e e k so leus 117 44 . P a r v a l b u m i n d i s t r i b u t i o n i n n o r m a l 2 - w e e k E D L 121 4 5 . P a r v a l b u m i n d i s t r i b u t i o n i n d y s t r o p h i c 2 - w e e k E D L 121 46 . M y o s i n A T P a s e o f n o r m a l 2 - w e e k E D L 123 v i i i 4 7 . M y o s i n A T P a s e o f d y s t r o p h i c 2 - w e e k E D L 123 4 8 . P a r v a l b u m i n l o c a l i z a t i o n i n n o r m a l 2 - w e e k so leus 126 49 . P a r v a l b u m i n l o c a l i z a t i o n i n d y s t r o p h i c 2 - w e e k so leus 126 50 . M y o s i n A T P a s e o f n o r m a l 2 - w e e k so leus 128 5 1 . M y o s i n A T P a s e o f d y s t r o p h i c 2 - w e e k so leus 128 52 . S t a n d a r d c u r v e f o r R I A o f a d u l t m u s c l e samp les 135 53 . S t a n d a r d c u r v e f o r R I A o f n e o n a t a l m u s c l e samp les 137 54. H i s t o g r a m o f R I A resu l ts f o r a d u l t samp les 139 55 . H i s t o g r a m o f R I A resu l ts o f neona ta l samp les 141 i x A C K N O W L E D G E M E N T A s t u d y s u c h as th is is n e v e r the w o r k o f o n e p e r s o n , a n d I t r u l y h a v e m a n y p e o p l e to w h o m I o w e m y g r a t i t u d e . F i r s t a n d f o r e m o s t I w i s h to e x t e n d m y a p p r e c i a t i o n to m y a d v i s o r D r . W i l l i a m O v a l l e w h o was d e t e r m i n e d that I r e c e i v e the best poss ib le a l l - r o u n d e d u c a t i o n w h i l e at U B C . I t h a n k h i m f o r h i s p a t i e n c e , l i b e r a l g u i d a n c e , p e r s i s t e n c e , a n d d e v o t e d in te res t i n m y e d u c a t i o n . I a m also d e e p l y i n d e b t e d to D r . K e n B a i m b r i d g e w h o t o o k m e u n d e r h i s w i n g , a l l o w e d m e to w o r k i n h i s l a b o r a t o r y , a n d t r i e d to m a k e m e a sc ien t i s t . T o the o the r m e m b e r s o f m y c o m m i t t e e , D r . W a y n e V o g l , D r . J o a n n e E m e r m a n , a n d to D r . D o w , t h a n k y o u f o r a l w a y s b e i n g there to h e l p w i t h m y p r o b l e m s no ma t te r h o w t r i v i a l . T o the late D r . J a s c h I o f f e r th i s w o r k as a t r i b u t e to h e r d e v o t i o n to the t ru ths o f s c i e n c e . I h o p e she w o u l d h a v e a p p r o v e d o f th is e x t e n s i o n o f h e r w o r k . I o w e a great d e a l to the t e c h n i c a l s t a f f w h o h a v e ass is ted m e t h r o u g h o u t m y s tud ies . I a m v e r y a p p r e c i a t i v e o f J e a n M c L e o d f o r h e r w o r k w i t h the a n i m a l s , p e r f o r m i n g the a m p u t a t i o n s a n d o v a r i a n t r ansp lan t s , a n d f o r i n t r o d u c i n g m e to s e v e r a l v a l u b l e t e c h n i q u e s . I w o u l d a lso l i k e to t h a n k H e l l a P r o c h a z s k a , Sue F i n l e y a n d B r u c e A n d e r s o n f o r t h e i r end less p a t i e n c e a n d i n s t r u c t i o n . A s p e c i a l t h a n k s goes to B e r n i e C o x f o r the r e p r o d u c t i o n o f m y o r i g i n a l p r i n t s . F i n a l l y , I w i s h to t h a n k m y co l l eagues a n d pee rs , b o t h i n a n d o u t s i d e o f the d e p a r t m e n t w h o h a v e m a d e these years o f s t u d y a t r u l y e n j o y a b l e e x p e r i e n c e . A n d las t , b u t a b o v e a l l no t the leas t , I t h a n k m y best f r i e n d a n d b igges t f a n , P a u l J o h n s o n . x D E D I C A T I O N T o A u n t i e B e e , w h o s p a r k e d m y in te res t i n the w o r l d o f s c i e n c e a n d i n the u n k n o w n . x i G E N E R A L I N T R O D U C T I O N M u s c u l a r d y s t r o p h y is a t e r m used to d e s c r i b e a g r o u p o f g e n e t i c d i s o r d e r s w h i c h are c h a r a c t e r i z e d b y p r o g r e s s i v e a n d severe d e g e n e r a t i o n o f s ke le ta l m u s c l e f i b e r s . T h e d isease a f f e c t s the m o r p h o l o g y , p h y s i o l o g y a n d b i o c h e m i c a l n a t u r e o f the m u s c l e f i b e r . A l t h o u g h n u m e r o u s a b n o r m a l i t i e s h a v e b e e n r e p o r t e d i n b o t h m u s c l e a n d n o n - m u s c l e t i ssue the p r i m a r y p r o d u c t o f the m u t a n t gene has no t b e e n i d e n t i f i e d . T h e recen t i s o l a t i o n o f a c a n d i d a t e f o r the c D N A ' s f o r a p o r t i o n o f the D u c h e n n e m u s c u l a r d y s t r o p h y gene appea rs to s u p p o r t the t h e o r y that the gene i n th is p a r t i c u l a r d i s o r d e r is i n v o l v e d i n the p r o d u c t i o n o f a m u s c l e p r o t e i n ( M o n a c o et a l . , 1986) . A n i m a l M o d e l s A n u m b e r o f a n i m a l m o d e l s h a v e b e e n u t i l i z e d i n m u s c u l a r d y s t r o p h y r e s e a r c h , a n d these i n c l u d e the h a m s t e r , c h i c k e n a n d m o u s e . I n th is thes is , the m u r i n e m o d e l f o r h e r e d i t a r y m u s c u l a r d y s t r o p h y w i l l o n l y be c o n s i d e r e d . E v e n t h o u g h no a n i m a l m o d e l c o m p l e t e l y r esemb les the h u m a n f o r m o f m u s c u l a r d y s t r o p h y , t h e y h a v e a d d e d an i n v a l u a b l e w e a l t h o f e x p e r i m e n t a l d a t a to o u r k n o w l e d g e o f the d isease a n d to the response o f m u s c l e i n a n a b n o r m a l e n v i r o n m e n t . T h e t w o mos t f r e q u e n t l y s t u d i e d m o u s e m o d e l s f o r p r o g r e s s i v e m u s c u l a r d y s t r o p h y are the 1 2 9 / R e J d y / d y ( M i c h e l s o n et a l . , 1955) a n d the C 5 7 B L / 6 J d y 2 J / d y 2 J ( M e i e r a n d S o u t h a r d , 1970) s t ra ins . M u s c u l a r d y s t r o p h y i n these t w o s t ra ins o f m o u s e is c a r r i e d b y a n a u t o s o m a l recess i ve gene ( H a r r i s a n d S l a t e r , 1980) . M i c e that are h o m o g e n e o u s at the d y s t r o p h i c a l le le a re c h a r a c t e r i z e d at a n e a r l y age o f 2 - 3 w e e k s b y s p l a y i n g a n d c l e n c h i n g o f the h i n d l i m b s , i n a b i l i t y to g rasp w i t h t h e i r h i n d fee t , a n d a d e c r e a s e d a b i l i t y to r e g a i n t he i r b a l a n c e . A s the d isease p rog resses , the h i n d l i m b s u n d e r g o severe a t r o p h y a n d b e c o m e a b n o r m a l l y e x t e n d e d a n d d y s f u n c t i o n a l . N o c l i n i c a l s igns are e v i d e n t i n the f o r e l i m b s . In m i c e h o m o z y g o u s f o r the d y 2 J a l l e le the d isease is e x p r e s s e d i n a m i l d e r f o r m t h a n that n o t e d i n m i c e o f the d y a l l e le . 1 M i c e o f the d y 2 J s t r a i n a re c h a r a c t e r i z e d b y a s l o w e r , p r o g r e s s i v e d e g e n e r a t i o n o f m u s c l e t i ssue. T h e d isease does no t a p p e a r to a f f e c t the m y o g e n i c p rocess s i nce m y o t u b e s are m o r p h o l o g i c a l l y n o r m a l a n d o n l y a f e w m y o f i b e r s are a l t e red at b i r t h ( P l a t z e r a n d P o w e l l , 1975) . R e c e n t l y B u l f i e l d et a l . (1984) d e s c r i b e d a n X - l i n k e d recess i ve m u t a n t i n the m o u s e ( m d x ) . A l t h o u g h a g e n e t i c h o m o l o g y to the h u m a n l ocus m a y e x i s t , a n d h i g h s e r u m e n z y m e l eve l s s i m i l a r to D u c h e n n e d y s t r o p h y h a v e b e e n r e p o r t e d , th is m u t a n t has s u b s e q u e n t l y b e e n c h a r a c t e r i z e d as a p o t e n t i a l m o d e l f o r m u s c l e r e g e n e r a t i o n . T h e m d x m o u s e s h o w s s igns o f m u s c l e f i b e r d e g e n e r a t i o n a n d a l t e r e d p h y s i o l o g i c a l responses u p to 3 - 4 w e e k s o f age . T h e r e is a d e c r e a s e d t w i t c h t e n s i o n , m u s c l e w e i g h t a n d a p r o l o n g e d h a l f - r e l a x a t i o n t i m e c o m p a r e d to the c o n t r o l a n i m a l . H o w e v e r , the m d x a d u l t m o u s e s h o w s n o s igns o f sus ta i ned m u s c l e w e a k n e s s , no c h a n g e s i n f i b e r t y p e d i f f e r e n t i a t i o n a n d measu res o f the p h y s i o l o g i c a l p a r a m e t e r s h a v e r e t u r n e d to n o r m a l . T h e r e f o r e , d u e to i ts h i g h r e g e n e r a t i v e c a p a c i t y the m d x m u t a n t has no t s e r v e d as a u s e f u l m o d e l f o r m u s c u l a r d y s t r o p h y . H i s t o l o g i c a l F e a t u r e s S e v e r a l h i s t o l o g i c a l a b n o r m a l i t i e s o c c u r i n the pos tna ta l a n d p r e - c l i n i c a l stages o f the m u r i n e d i sease , i n d i c a t i n g that i ts p h e n o t y p i c e x p r e s s i o n o c c u r s e a r l y i n m u s c l e d e v e l o p m e n t ( P l a t z e r a n d P o w e l l , 1975; M e n d e l l et a l . , 1979; S u m m e r s a n d P a r s o n s , 1981b) . T h e c h a r a c t e r i s t i c f ea tu re o f d y s t r o p h i c m o u s e m u s c l e is the h e t e r o g e n e i t y i n a p p e a r a n c e o f e x t r a f u s a l f i b e r s . A t e a r l y o r la te r stages o f the d i sease , m u s c l e f i b e r s o f v a r i a b l e s i ze a n d those that are n e c r o t i c , r e g e n e r a t i n g o r s p l i t t i n g are c o m m o n l y o b s e r v e d i n a g i v e n f a s c i c l e n e x t to those w i t h a n o r m a l a p p e a r a n c e ( C u l l e n a n d M a s t a g l i a , 1980) . F r a g m e n t a t i o n o f m u s c l e f i b e r s is c o m m o n i n the i n i t i a l s tages, as a re s igns o f a b o r t i v e r e g e n e r a t i o n ( S u m m e r s a n d P a r s o n s , 1981b) . D u r i n g the la ter phases o f the d i s o r d e r there is a s i g n i f i c a n t p r o l i f e r a t i o n o f e n d o m y s i a l c o n n e c t i v e t issue w h i c h m a y i m p a i r m u s c l e f i b e r r e c o v e r y ( M e n d e l l et a l . , 1979) . 2 A c h a r a c t e r i s t i c h i s t o l o g i c a l f ea tu re o f m u r i n e d y s t r o p h y is the p resence o f r e g e n e r a t i v e a c t i v i t y at s o m e d i s t ance f r o m the a rea o f f i b e r nec ros i s ( B a n k e r et a l . , 1979; S u m m e r s a n d P a r s o n s , 1981b) . T h i s f e a t u r e has b e e n a t t r i b u t e d to a n a b n o r m a l response o f m y o s a t e l l i t e ce l l s to the even ts o f f i b e r nec ros i s ( S u m m e r s a n d P a r s o n s , 1981a) . M y o s a t e l l i t e ce l l s f use to f o r m n e w m y o t u b e s . T h e s e i n v e s t i g a t o r s r e p o r t e d that d y s t r o p h i c m o u s e m u s c l e has a n adequa te n u m b e r o f sa te l l i te ce l l s a n d , t h e r e f o r e , i t is the s u b s e q u e n t d e v e l o p m e n t o f the m y o t u b e s w h i c h is a b n o r m a l a n d not t he i r i n i t i a l f o r m a t i o n . T h e m y o s a t e l l i t e c e l l p o p u l a t i o n is h i g h e r i n d y s t r o p h i c m u s c l e t han i n n o r m a l m u s c l e a n d decreases w i t h age ( O n t e l l et a l . , 1984) . T h i s f a c t o r was sugges ted to be r e s p o n s i b l e f o r the i n a b i l i t y o f the r e g e n e r a t i v e response o f d y s t r o p h i c m u s c l e to k e e p p a c e w i t h the m u s c l e d e t e r i o r a t i o n . O n e o f the f i r s t u l t r a s t r u c t u r a l a l t e ra t i ons i n d y s t r o p h i c m o u s e m u s c l e is d i l a t a t i o n o f the S R , i n c l u d i n g i ts n e t w o r k o f l o n g i t u d i n a l t u b u l e s a n d t e r m i n a l c i s te rnae ( P l a t z e r a n d P o w e l l , 1975) . S u b s e q u e n t l y , m i t o c h o n d r i a m a y a p p e a r s w o l l e n a n d f i l l e d w i t h m a t r i x g r a n u l e s , a n d m y o f i b r i l s b e c o m e f r a g m e n t e d a n d a t r o p h i e d . R e t r a c t i o n c lo ts f o r m a n d the s a r c o l e m m a b e c o m e s s l i g h t l y f r a g m e n t e d , e v e n t u a l l y l e a d i n g to c o a g u l a t i o n nec ros i s a n d p h a g o c y t i c i n f i l t r a t i o n . U l t r a s t r u c t u r a l s tud ies o f m o t o r e n d - p l a t e s i n d y s t r o p h i c m i c e ( B a n k e r et a l . , 1979) h a v e i n d i c a t e d the p resence o f b o t h n e u r a l a n d m u s c u l a r a b n o r m a l i t i e s assoc ia ted w i t h the d isease i n th is a n i m a l m o d e l . A l t h o u g h no d e f i n i t e m y o t r o p h i c i n f l u e n c e s o n n e r v e h a v e b e e n o b s e r v e d i n the d y 2 J s t r a i n , a b n o r m a l n e u r o t r o p h i c i n f l u e n c e s because o f the d e g e n e r a t i o n o f the m o t o r n e u r o n s h a v e b e e n d o c u m e n t e d (Sai to et a l . , 1983) . P r e s y n a p t i c a n d p o s t s y n a p t i c a l t e r a t i o n s , s u c h as s i m p l i f i c a t i o n a n d a t r o p h y o f the j u n c t i o n a l f o l d s a n d r e t r a c t i o n o f the n e r v e t e r m i n a l s f r o m the p o s t s y n a p t i c m e m b r a n e , h a v e b e e n d e s c r i b e d . - A s w e l l , s i g n i f i c a n t r e d u c t i o n s i n the n u m b e r o f m y e l i n a t e d a x o n s have b e e n r e p o r t e d i n b o t h a l l e l i c s t ra ins ( M o n t g o m e r y a n d S w e n a r c h u k , 1978) . I n the d y 2 J m u t a n t f a i l u r e o f the S c h w a n n c e l l to enshea th the n a k e d a x o n s o f s p i n a l roots o c c u r s e a r l y i n pos tna ta l d e v e l o p m e n t ( B r a y et a l . , 1978) . T h i s g r o u p c o n c l u d e d that the 3 S c h w a n n c e l l d e f i c i t was a d e v e l o p m e n t a l a b n o r m a l i t y assoc ia ted w i t h a neona ta l i m p a i r m e n t o f S c h w a n n c e l l m u l t i p l i c a t i o n . In a d d i t i o n , d i s c o n t i n u i t i e s i n the basa l l a m i n a s o f S c h w a n n ce l l s is a c o m m o n a b n o r m a l i t y o b s e r v e d i n the d y 2 J a d u l t d y s t r o p h i c m o u s e ( M a d r i d et a l . , 1975) . F i b e r T y p e s E x t r a f u s a l m u s c l e f i b e r s a re c l a s s i f i e d i n t o t w o m a j o r t y p e s , t y p e 2 ( fast ) a n d t y p e 1 ( s l o w ) , a c c o r d i n g to m y o s i n A T P a s e o r g l y c o l y t i c e n z y m e s ta ins ( B r o o k e a n d K a i s e r , 1970; P e t e r et a l . , 1972) . T y p e 1 m u s c l e f i b e r s h a v e l o w m y o s i n A T P a s e a c t i v i t y ( p H 9 .4 ) , b u t a re a c i d s tab le a c c o r d i n g to c o n v e n t i o n a l h i s t o c h e m i c a l s t a i n i n g p r o c e d u r e s ( B r o o k e a n d K a i s e r , 1970) . T h e s e f i b e r s a re u s u a l l y assoc ia ted w i t h s l o w c o n t r a c t i o n t i m e s , a p o o r l y d e v e l o p e d g l y c o l y t i c e n z y m e s y s t e m , a h i g h m i t o c h o n d r i a l c o n t e n t , a n d a h i g h p o t e n t i a l f o r o x i d a t i v e e n z y m e a c t i v i t i e s . C o n s e q u e n t l y , t y p e 1 f i b e r s a re a lso t e r m e d s l o w - o x i d a t i v e (SO) f i b e r s (Pe te r et a l . , 1972) . C o n v e r s e l y , t y p e 2 e x t r a f u s a l f i b e r s h a v e h i g h m y o s i n A T P a s e a c t i v i t y , fas t c o n t r a c t i o n t i m e s , a n d a w e l l d e v e l o p e d g l y c o l y t i c e n z y m e s y s t e m . O n the o the r h a n d , these f i b e r s are c h a r a c t e r i z e d b y a l o w m i t o c h o n d r i a l c o n t e n t a n d p o o r o x i d a t i v e e n z y m e a c t i v i t y . T y p e 2 f i b e r s m a y be d i v i d e d i n t o s e v e r a l s u b g r o u p s ( types 2 A , 2 B , a n d 2 C ) o n the basis o f d i f f e r e n t i a l s u s c e p t i b i l i t y to t r ea tmen t w i t h p r e i n c u b a t i o n b u f f e r s at v a r y i n g p H va lues ( B r o o k e a n d K a i s e r , 1970) . T y p e 2 A f i b e r s a re m o r e a c i d - l a b i l e t h a n f i b e r s o f t y p e 2 B , w h e r e a s t y p e 2 C f i b e r s a re s tab le at a c i d a n d a l k a l i n e p r e i n c u b a t i o n . G e n e r a l l y , t y p e 2 A f i b e r s are a lso t e r m e d f a s t - o x i d a t i v e - g l y c o l y t i c ( F O G ) w h i l e t ype 2 B f i b e r s a re d e s i g n a t e d f a s t - g l y c o l y t i c ( F G ) a c c o r d i n g to t h e i r o x i d a t i v e a n d g l y c o l y t i c e n z y m e sys tems (Pe te r et a l . , 1972) . T h e t w o n o m e n c l a t u r e s are o f t e n used i n t e r c h a n g e a b l y ; h o w e v e r , th is c o r r e l a t i o n does no t a l w a y s a p p l y to t y p e 2 A a n d 2 B f i b e r s o f a l l spec ies ( G r e e n et a l . , 1982) . It i s , t h e r e f o r e , m o r e a p p r o p r i a t e to u t i l i z e c o m b i n e d t e r m i n o l o g y i n r e f e r r i n g to e x t r a f u s a l f i b e r s s u c h as t y p e 2 A / F O G o r t ype 2 A / F G . 4 F i b e r t y p e s u s c e p t i b i l i t y has b e e n r e p o r t e d to d i f f e r b e t w e e n the t w o d y s t ra ins o f d y s t r o p h i c m o u s e . I n the d y / d y s t r a i n , the f a s t - t w i t c h m u s c l e s are p r e f e r e n t i a l l y i n v o l v e d i n the e a r i y phases o f the d isease ( B u t l e r a n d C o s m o s , 1977) , w h e r e a s m u s c l e s c o m p o s e d o f p r e d o m i n a n t l y s l o w - t w i t c h o x i d a t i v e f i b e r s a re t h o u g h t to be m o r e s e v e r e l y a f f e c t e d i n the d y 2 J / d y 2 J s t r a i n ( D r i b i n a n d S i m p s o n , 1977) . A n u m b e r o f repo r t s p r o v i d e e v i d e n c e that m u r i n e m u s c u l a r d y s t r o p h y c a n a f f e c t the h i s t o c h e m i c a l c h a r a c t e r o f the e x t r a f u s a l f i b e r t ypes i n s k e l e t a l m u s c l e ( B u t l e r a n d C o s m o s , 1977; D r i b i n a n d S i m p s o n , 1977; S i l v e r m a n a n d A t w o o d , 1980; P a r r y a n d P a r s l o w , 1981 ; O v a l l e et a l . , 1983 ; W i r t z et a l . , 1983) . It has r e c e n t l y b e e n sugges ted tha t these c h a n g e s i n v o l v e a c o n v e r s i o n to a n ' i n t e r m e d i a t e ' t y p e o f f i b e r w i t h i m m a t u r e h i s t o c h e m i c a l p r o p e r t i e s ( W i r t z et a l . , 1983) , o r d u e to a r e t a r d a t i o n i n t h e i r n o r m a l pos tna ta l d e v e l o p m e n t ( O v a l l e et a l . , 1983) . S tud ies o f d y s t r o p h i c m o u s e m u s c l e d u r i n g e a r l y stages o f the d isease ( P a r r y a n d P a r s l o w , 1981 ; W i r t z et a l . , 1983) p o i n t to a d r a m a t i c dec rease i n e x t r a f u s a l f i b e r s o f the t y p e 2 / F G v a r i e t y i n f a s t - t w i t c h m u s c l e s . D u r i n g pos tna ta l d e v e l o p m e n t o f the s l o w - t w i t c h so leus i n d y s t r o p h i c m i c e , an i nc rease i n t y p e 2 / F O G e x t r a f u s a l f i b e r s a n d a c o n c o m i t a n t dec rease i n the t y p e 1 / S O f i b e r s h a v e a lso b e e n r e p o r t e d ( O v a l l e et a l . , 1983) . B o t h the p e r c e n t a g e o f F O G f i b e r s a n d a b n o r m a l (or a t y p i c a l ) f i b e r s w h i c h are h i s t o c h e m i c a l l y s i m i l a r to t y p e 2 C f i b e r s , s i g n i f i c a n t l y i n c r e a s e w i t h a d v a n c e m e n t o f the d i sease . T h i s c y t o c h e m i c a l t r e n d i n e x t r a f u s a l f i b e r t ypes i n m u r i n e d y s t r o p h y is a lso s u p p o r t e d b y p h y s i o l o g i c a l ( B r e s s l e r et a l . , 1983) a n d b i o c h e m i c a l ( Jasch et a l . , 1982) d a t a a n d is f u r t h e r c o m p l e m e n t e d b y i n v e s t i g a t i o n s that p o i n t to the a p p e a r a n c e o f a f e t a l f o r m o f m y o s i n i n a d u l t d y s t r o p h i c h u m a n m u s c l e ( F i t z s i m o n s a n d H o h , 1981) a n d a d u l t c h i c k e n d y s t r o p h i c m u s c l e ( B a n d m a n , 1985) . C o n v e r s i o n o f e x t r a f u s a l f i b e r t ypes to those r e s e m b l i n g a t y p e 2 C f i b e r m a y i n d i c a t e a n a l t e r a t i o n i n the m a t u r a t i o n a l p rocess o f s ke le ta l m u s c l e i n m u r i n e d y s t r o p h y ( O v a l l e et a l . , 1983; W i r t z et a l . , 1983) . L e s s is k n o w n a b o u t the e f f e c t s o f d y s t r o p h y o n the h i s t o c h e m i c a l d e v e l o p m e n t o f i n t r a f u s a l f i b e r s i n the m u s c l e s p i n d l e . T h e s e s p e c i a l i z e d a n d p a r t i a l l y e n c a p s u l a t e d 5 s e n s o r y s t r e t ch recep to r s a p p e a r r e l a t i v e l y res is tan t to the d isease ( S w a s h , 1982) . M o r p h o l o g i c a l l y a b n o r m a l i n t r a f u s a l f i b e r s h a v e b e e n n o t e d i n h u m a n D u c h e n n e d y s t r o p h y ( S w a s h a n d F o x , 1976) . L i g h t m i c r o s c o p e s tud ies o f m u s c l e s p i n d l e s i n m u r i n e d y s t r o p h y are l i m i t e d a n d h a v e no t d e t e c t e d a n y a b n o r m a l i t i e s e i t h e r i n i n t r a f u s a l f i b e r m o r p h o l o g y ( M e i e r , 1969) o r h i s t o c h e m i s t r y ( Y e l l i n , 1974b) . A recen t r e p o r t ( O v a l l e a n d D o w , 1986) has s h o w n s o m e a l te ra t i ons i n s p i n d l e m o r p h o l o g y i n t e r m i n a l stages o f m u r i n e m u s c u l a r d y s t r o p h y . T h e s e s tud ies dea l t w i t h e i t h e r the m o r p h o l o g y o r h i s t o c h e m i s t r y o f the i n t r a f u s a l f i b e r s i n a c o n f i n e d r e g i o n o f the s p i n d l e . I n a d d i t i o n , the n o r m a l t i m e c o u r s e o f i n t r a f u s a l f i b e r t y p e d e v e l o p m e n t i n d y s t r o p h i c m u s c l e has no t b e e n c o n s i d e r e d . S i n c e a c c u r a t e i d e n t i f i c a t i o n o f i n t r a f u s a l f i b e r t ypes b y m y o s i n A T P a s e s t a i n i n g m e t h o d s r e q u i r e s that s e r i a l sec t i ons be cu t t h r o u g h o u t the l e n g t h o f a n i n d i v i d u a l s p i n d l e ( S o u k u p , 1976) , f u r t h e r i n v e s t i g a t i o n s o f th is t y p e are necessa ry to d e t e r m i n e the r e s p o n s e , i f a n y , o f i n t r a f u s a l f i b e r s to d y s t r o p h y . P h y s i o l o g i c a l a n d B i o c h e m i c a l C h a n g e s T h e resu l ts o f s tud ies o f the m e c h a n i c a l p r o p e r t i e s o f s k e l e t a l m u s c l e i n m u r i n e d y s t r o p h y are cons i s ten t w i t h the h i s t o c h e m i c a l f i n d i n g s . G e n e r a l l y , the re is a dec rease i n b o t h t w i t c h a n d te tan i c f o r c e , a n i nc rease c o n t r a c t i o n t i m e a n d t i m e - t o - p e a k t e n s i o n a n d a p r o l o n g a t i o n i n the h a l f - r e l a x a t i o n t i m e o f f a s t - t w i t c h m u s c l e ( P a r s l o w a n d P a r r y , 1981 ; B r e s s l e r et a l . , 1983 ; P a r r y a n d D e s y p r i s , 1983) . F o r the d y s t r o p h i c s o l e u s , d e c r e a s e d t w i t c h a n d te tanus t e n s i o n has b e e n o b s e r v e d i n m i c e f r o m 4 to 32 w e e k s o f age (B ress le r et a l . , 1983) . H o w e v e r , the p r o l o n g a t i o n o b s e r v e d i n the t i m e - t o - p e a k t e n s i o n a n d h a l f - r e l a x a t i o n t i m e i n the y o u n g d y s t r o p h i c so leus was no t s i g n i f i c a n t l y d i f f e r e n t f r o m the n o r m a l va lues b y 32 w e e k s o f age . W h i l e the p r o l o n g e d t i m e - t o - p e a k t e n s i o n i n the d y s t r o p h i c f a s t - t w i t c h m u s c l e has b e e n f o u n d to c o r r e l a t e w i t h the p r e s e n c e o f s l o w m y o s i n , the p r o l o n g e d t w i t c h r e l a x a t i o n phase seen i n these m u s c l e s has no t ( P a r r y a n d D e s y p r i s , 1983) . T h e s e changes suggest a 6 deficit in both the activation of dystrophic muscle and in the relaxation phases of muscle contraction. An altered excitation-contraction coupling in murine dystrophic muscle may be associated with a decreased rate of removal of calcium ions from the myoplasm during muscle contraction (Mrak, 1985). A deficiency in the calcium transport system may in turn be due to an abnormality in the handling of calcium ions by the sarcoplasmic reticulum or the calcium-binding proteins, to an altered signal propagation by the transverse tubules, to an abnormality in the muscle membrane surface, or to a lack of neurotrophic factor. Biochemically, dystrophic mouse skeletal muscle is characterized by a decrease in the uptake and rate of C a 2 + transport (Sreter et al., 1966; Mrak and Baskin, 1978; Wood et al., 1978), a decrease in maximum steady-state concentration of phosphoenzyme intermediate, and a decrease in Ca2+-sensitive ATPase activity (Mrak and Baskin, 1978; Martonosi, 1982). In addition, densitometric analysis of dystrophic SR preparations indicate a decrease in calsequestrin in the dy 2 J mutant (Butcher and Tomkins, 1986). Calcium functions as an intracellular messenger in many cellular processes from muscle contraction to hormone secretion (Carafoli and Penniston, 1985). Regulation of calcium is mediated by certain proteins that interact with the ion governing its transmission, reception and removal, thereby controlling its concentration in the intracellular milieu. The roles of calcium, and its affiliated binding proteins (calmodulin, myosin phosphorylated light-chain, parvalbumin, and troponin C) in muscle contraction have been investigated extensively (reviewed by Carafoli and Penniston, 1985). Binding of calcium to troponin C (Tn-C) causes the muscle to contract, and binding of calcium to calmodulin initiates an important enzyme reaction in a process that produces ATP. Parvalbumin, on the other hand, is thought to act as a calcium buffer and a soluble relaxing factor in fast-twitch muscle (Pechere et al., 1977; Haiech et al., 1979a). A disturbance of calcium homeostasis in skeletal muscle fibers of 7 d y s t r o p h i c m i c e has b e e n l i n k e d to a n e l e v a t e d s a r c o p l a s m i c C a 2 + c o n c e n t r a t i o n , p o s s i b l y d u e to p e r m e a b i l i t y changes i n the s a r c o l e m m a ( D u n c a n , 1978) . E v e n t h o u g h the c a l c i u m u p t a k e a b i l i t y o f the S R has d e c r e a s e d , c a l c i u m re lease f r o m the S R m a y be u n a f f e c t e d i n m u r i n e d y s t r o p h y ( V o l p e et a l . , 1984) . T h i s w o u l d i n d i c a t e that the p r o b l e m l ies i n the c a l c i u m r e g u l a t i o n s y s t e m d u r i n g the r e l a x a t i o n phase a n d not the i n i t i a t i o n o f c o n t r a c t i o n . I n l i gh t o f the p r o p o s e d f u n c t i o n s o f P V i n f a s t - t w i t c h m u s c l e , a n a l t e r a t i o n i n the f u n c t i o n a l c a p a c i t y o f th is p r o t e i n m a y be o f s i g n i f i c a n c e i n the r e g u l a t i o n o f C a 2 + i n the d i seased m u s c l e ( K l u g et a l . , 1985) . H e n c e , P V has b e c o m e a t o p i c o f i n te res t i n C a 2 + - r e g u l a t e d p rocesses s u c h as those o c c u r r i n g d u r i n g m u s c l e c o n t r a c t i o n a n d i n m y o p a t h i e s ( C e l i o a n d H e i z m a n n , 1982; S t u h l f a u t h et a l . , 1984) . I n m u r i n e d y s t r o p h y , the P V c o n t e n t o f s o m e f a s t - t w i t c h m u s c l e s is s i g n i f i c a n t l y r e d u c e d i n the la te r stages o f the d isease ( Jasch a n d M o a s e , 1985; Pe t te et a l . , 1985 ; J o h n s o n a n d O v a l l e , 1986) . T h e ex ten t to w h i c h th i s d e c r e a s e d c o n c e n t r a t i o n o f P V a f f e c t s the b u f f e r i n g c a p a c i t y o f i n t r a c e l l u l a r C a 2 + i n d y s t r o p h i c m u s c l e is u n c l e a r . T h e e t i o l o g y o f m u r i n e m u s c u l a r d y s t r o p h y is u n k n o w n , b u t e v i d e n c e p o i n t i n g to a n a l t e r e d m e m b r a n e s t r u c t u r e o r f u n c t i o n has b e e n a c c u m u l a t i n g . E x t r a c e l l u l a r t racers f o c a l l y pene t ra te the s a r c o l e m m a o f m u s c l e f i b e r s i n d y s t r o p h i c m u r i n e m u s c l e ( B a n k e r et a l . , 1979; M e n d e l l et a l . , 1979) i n d i c a t i n g that m e m b r a n e i n t e g r i t y is d i s r u p t e d . L e a k a g e o f c a l c i u m i n t o m u s c l e f i b e r s o c c u r s d u r i n g e a r l y c e l l u l a r nec ros i s s u g g e s t i n g that f o c a l d e f e c t s i n the p l a s m a m e m b r a n e d o no t r ep resen t the i n i t i a l even t i n c e l l u l a r d e g e n e r a t i o n ( M e n d e l l et a l . , 1979) . A b n o r m a l p r o p e r t i e s a p p l y to the i n t e r n a l m e m b r a n e sys tems o f d y s t r o p h i c m u s c l e as w e l l . In a d d i t i o n to the u l t r a s t r u c t u r a l s tud ies o f the S R i n m o u s e d y s t r o p h y ( P l a t z e r a n d P o w e l l , 1975) f r e e z e - f r a c t u r e images h a v e b e e n p u b l i s h e d s h o w i n g S R d i l a t a t i o n a n d f r a g m e n t a t i o n w i t h n o a b n o r m a l i t i e s o f the t ransverse t ubu les ( M r a k a n d B a s k i n , 1978) . A c o r r e l a t i o n o f the m o r p h o l o g i c a l , b i o c h e m i c a l a n d p h y s i o l o g i c a l d a t a o n d y s t r o p h i c m u s c l e is d i f f i c u l t because o f a c o m b i n a t i o n o f f a c t o r s that m a y c o n t r i b u t e 8 to fa l se i m p r e s s i o n s . I n t e r p r e t a t i o n o f the d a t a is c o m p l i c a t e d a n d l i m i t e d b y changes i n f i b e r t y p e c o m p o s i t i o n , spec ies v a r i a t i o n s , a n d s a m p l i n g p r o b l e m s d u e to the p a r t i c u l a r stage o f the d isease . B i o c h e m i c a l p r e p a r a t i o n s o f h o m o g e n i z e d t issue are h i n d e r e d b y the p r e s e n c e o f i n c r e a s e d a m o u n t s o f f i b r o u s a n d f a t t y c o n n e c t i v e - t i s s u e , p ro tease a c t i v i t y , a n d the re lease o f l y s o s o m a l e n z y m e s d u r i n g p r e p a r a t i o n . A s w e l l , the p o s s i b i l i t y tha t some o f the o b s e r v e d b i o c h e m i c a l a l t e ra t i ons c o u l d be s e c o n d a r y r eac t i ons to a n u n e x p o s e d p r i m a r y d e f e c t m u s t be c o n s i d e r e d ( M a r t o n o s i , 1982; E n g e l , 1986) . S ta temen t o f the O b j e c t i v e s I h a v e d i v i d e d th is p r o j e c t i n t o t w o s tud ies . F i r s t l y , I w i s h e d to s t u d y the m o r p h o l o g i c a l , h i s t o c h e m i c a l a n d q u a n t i t a t i v e changes i n m u s c l e s p i n d l e s a n d t he i r i n t r a f u s a l f i b e r s i n fas t a n d s l o w - t w i t c h m u s c l e s d u r i n g the n e o n a t a l stages o f m u r i n e m u s c u l a r d y s t r o p h y . T h i s w o r k has r e c e n t l y b e e n p u b l i s h e d ( J o h n s o n a n d O v a l l e , 1986) . S e c o n d l y , I w i s h e d to test the h y p o t h e s i s that d i f f e r e n c e s i n the p r o f i l e o f c e r t a i n p r o t e i n s i so l a t ed f r o m d y s t r o p h i c m u s c l e r e f l e c t a loss o r a l t e r a t i o n o f m u s c l e t y p e d i f f e r e n t i a t i o n . T h i s was a t t e m p t e d b y d e t e r m i n i n g i f the p h y s i o l o g i c a l response o f fas t a n d s l o w - t w i t c h m u s c l e i n the d y 2 J m o u s e , w i t h respec t to h a l f - r e l a x a t i o n t i m e , a n d the a c c o m p a n y i n g changes i n e x t r a f u s a l f i b e r t y p e e x p r e s s i o n are assoc ia ted w i t h a n a l t e r a t i o n i n the c a l c i u m - b i n d i n g p r o t e i n p a r v a l b u m i n . A n a l t e r a t i o n i n p a r v a l b u m i n m a y a lso be r e s p o n s i b l e f o r a d i s t u r b a n c e i n the c a l c i u m homeos tas i s o f d y s t r o p h i c m u s c l e . A s w e l l , I w i s h e d to d e t e r m i n e w h e t h e r p a r v a l b u m i n as an i m m u n o c h e m i c a l m a r k e r c o u l d be used to de tec t the e x p r e s s i o n o f d y s t r o p h y i n m u s c l e f i b e r s at a n e a r l i e r stage t h a n m y o s i n A T P a s e h i s t o c h e m i s t r y . A m o r e e x t e n s i v e r e v i e w o f the m u s c l e s p i n d l e a n d o f p a r v a l b u m i n is g i v e n i n the i n t r o d u c t i o n to the c o r r e s p o n d i n g c h a p t e r s . . 9 C H A P T E R 1. P o s t n a t a l d e v e l o p m e n t o f m u s c l e s p i n d l e s i n g e n e t i c a l l y d y s t r o p h i c m i c e I N T R O D U C T I O N M a m m a l i a n m u s c l e s p i n d l e s are s e n s o r y s t r e t ch r e c e p t o r s tha t c o n t a i n e n c a p s u l a t e d i n t r a f u s a l m u s c l e f i b e r s . T h e s e f i b e r s c a n be d i f f e r e n t i a t e d i n t o th ree t ypes a n d h a v e b e e n d e s i g n a t e d b a g x , b a g 2 a n d c h a i n f i b e r s a c c o r d i n g to t he i r m o r p h o l o g i c a l ( B a n k s et a l . , 1977) , h i s t o c h e m i c a l ( O v a l l e a n d S m i t h , 1972) , a n d p h y s i o l o g i c a l ( K e n n e d y et a l . , 1980; B o y d a n d S m i t h , 1984) f ea tu res . I n con t ras t to t h e i r n e i g h b o u r i n g e x t r a f u s a l f i b e r s , m a m m a l i a n i n t r a f u s a l f i b e r s v a r y h i s t o c h e m i c a l l y a l o n g t he i r i n d i v i d u a l l eng ths ( Y e l l i n , 1974a ; B a n k s et a l . , 1977; K u c e r a , 1977; K u c e r a a n d D o r o v i n i - z i s , 1979; S o u k u p et a l . , 1979;) . I m m u n o h i s t o c h e m i c a l s tud ies i n d i c a t e that the m y o s i n c o m p o s i t i o n o f the i n t r a f u s a l f i b e r s d i f f e r s b e t w e e n b a g a n d c h a i n f i b e r s ( P i e r o b o n B o r m i o l i et a l . , 1980; et K r o n n i e et a l . , 1981) . U n l i k e the e x t r a f u s a l f i b e r s , w h i c h are m o r p h o l o g i c a l l y a l t e r e d i n h u m a n ( C u l l e n a n d M a s t a g l i a , 1980) a n d m u r i n e ( L a w et a l . , 1983) m u s c u l a r d y s t r o p h y , s p i n d l e s a n d t h e i r i n t r a f u s a l f i b e r s a p p e a r to be r e l a t i v e l y res is tan t to the d isease ( S w a s h , 1982). A l t h o u g h a b n o r m a l i n t r a f u s a l f i b e r s h a v e b e e n r e p o r t e d i n m y o t o n i a ( S w a s h a n d F o x , 1975 ; M a y n a r d , et a l . , 1977) a n d D u c h e n n e d y s t r o p h y ( S w a s h a n d F o x , 1976) i n h u m a n s , s tud ies o f s p i n d l e m o r p h o l o g y i n m u r i n e d y s t r o p h y ( M e i e r , 1969; Y e l l i n , 1974b ; J a m e s a n d M e e k , 1979) h a v e f a i l e d to s h o w a n y p a t h o l o g i c a l c h a n g e s assoc ia ted w i t h the d i sease . T h e s e s tud ies e i t he r dea l t w i t h e i t he r the m o r p h o l o g y o r h i s t o c h e m i s t r y o f the i n t r a f u s a l f i b e r s i n a c o n f i n e d r e g i o n o f the s p i n d l e . I n a d d i t i o n , the n o r m a l t i m e c o u r s e o f i n t r a f u s a l f i b e r t y p e d e v e l o p m e n t i n d y s t r o p h i c m u s c l e has not b e e n c o n s i d e r e d . D e s p i t e the e x t e n s i v e l i t e ra tu re p u b l i s h e d o n m u s c u l a r d y s t r o p h y , i n f o r m a t i o n c o n c e r n i n g the e a r l y d e v e l o p m e n t o f the d isease w i t h respec t to m u s c l e s p i n d l e s is 10 lacking. Thus, the present study was undertaken to elucidate whether intrafusal fiber type alterations could be detected in dystrophic animals during initial stages of the disease. This study, therefore, assesses the early postnatal development of muscle spindles in dystrophic mice and compares their distribution and morphology in selected fast and slow-twitch muscles of normal age-matched controls. 11 M A T E R I A L S A N D M E T H O D S N o r m a l C 5 7 B L / 6 J ( + / + ) a n d d y s t r o p h i c C 5 7 B L / 6 J ( d y 2 J / d y 2 J ) m i c e w e r e h o u s e d i n the m o u s e c o l o n y m a i n t a i n e d i n o u r d e p a r t m e n t . T h e o r i g i n a l b r e e d i n g pa i r s o f b o t h the n o r m a l a n d d y s t r o p h i c s t r a i n w e r e o b t a i n e d f r o m J a c k s o n L a b o r a t o r i e s , B a r H a r b o u r , M E . M a l e h o m o z y g o u s o f f s p r i n g w e r e se lec ted at pos tna ta l ages o f 1, 2 , a n d 3 w e e k s . W h e n p o s s i b l e , n o r m a l c o n t r o l s o f s i m i l a r ages w e r e s a m p l e d at the same t i m e as t h e i r d y s t r o p h i c c o u n t e r p a r t s . A to ta l o f 28 m u s c l e s f r o m 14 n o r m a l m i c e a n d 30 m u s c l e s f r o m 15 d y s t r o p h i c m i c e w e r e e x a m i n e d . H i s t o c h e m i s t r y A n i m a l s w e r e k i l l e d b y e i t he r c e r v i c a l d i s l o c a t i o n o r d e c a p i t a t i o n . T h e r i gh t e x t e n s o r d i g i t o r u m l ongus ( E D L ) a n d so leus w e r e e x p o s e d , s u p e r f i c i a l f a s c i a r e m o v e d a n d t h e n q u i c k l y e x c i s e d . E a c h m u s c l e was p l a c e d o n s u r g i c a l g a u z e m o i s t e n e d b y p h y s i o l o g i c a l s a l i n e a n d w e t m u s c l e w e i g h t s w e r e m e a s u r e d . M u s c l e s w e r e e m b e d d e d i n f r e s h m o u s e l i v e r , t a k i n g ca re to no te the l o c a t i o n o f the t e n d o n s f o r f u t u r e o r i e n t a t i o n . T h e s p e c i m e n s w e r e m o u n t e d o n c h u c k s u s i n g g u m t r a g a c a n t h a n d q u i c k l y f r o z e n i n i s o p e n t a n e c o o l e d w i t h l i q u i d n i t r o g e n . T h e c h u c k s w e r e a l l o w e d to e q u i l i b r a t e to - 2 0 ° C i n the c r yos ta t f o r 1 h o u r p r i o r to s e c t i o n i n g . M u s c l e s w e r e s e r i a l l y s e c t i o n e d ( 1 0 p m t h i c k ) f r o m o r i g i n to i n s e r t i o n , a n d sec t i ons w e r e c o l l e c t e d o n glass c o v e r s l i p s . A t e v e r y 1 0 0 p m , th ree c o n s e c u t i v e t ransve rse sec t i ons w e r e m o u n t e d o n separa te c o v e r s l i p s f o r m y o s i n A T P a s e h i s t o c h e m i s t r y at a c i d ( p H 4.2 a n d p H 4.6) a n d a l k a l i n e ( p H 9.4) p r e i n c u b a t i o n s ( D u b o w i t z a n d B r o o k e , 1973) . T h i s sequence was f o l l o w e d t h r o u g h o u t the l e n g t h o f e a c h m u s c l e , a n d the d i s t r i b u t i o n o f m u s c l e s p i n d l e s was m a p p e d o v e r t h e i r f u l l ex ten t at a c i d a n d a l k a l i n e p r e i n c u b a t i o n s u s i n g the l i g h t m i c r o s c o p e 4 0 x o b j e c t i v e . I n t r a f u s a l f i b e r s w e r e t y p e d a c c o r d i n g to the b a g l 5 b a g 2 a n d c h a i n c l a s s i f i c a t i o n ( O v a l l e a n d S m i t h , 1972) . T h e h i s t o c h e m i c a l p r o f i l e f o r e a c h i n t r a f u s a l f i b e r was t h e n e s t a b l i s h e d t h r o u g h o u t i ts l e n g t h , e x c e p t f o r the e q u a t o r i a l 12 z o n e , w h e r e l i t t l e o r no s t a i n i n g o c c u r r e d . In a d d i t i o n , m o r p h o l o g i c a l c r i t e r i a used to d i s t i n g u i s h c h a i n f r o m b a g f i b e r s a n d b a g j f r o m b a g 2 f i b e r s i n c l u d e d c r o s s - s e c t i o n a l d i a m e t e r s a n d f i b e r l eng ths . I n . mos t cases , c h a i n f i b e r s w e r e s m a l l e r t h a n b a g f i b e r s ; h o w e v e r , those that w e r e s i m i l a r i n s i ze to b a g f i b e r s e x h i b i t e d the s t a i n i n g p r o p e r t i e s o f s m a l l e r c h a i n f i b e r s . Q u a n t i t a t i o n M u s c l e s p i n d l e s , o r n o n e n c a p s u l a t e d i n t r a f u s a l ' f i b e r s , w e r e l o c a t e d u n d e r the m i c r o s c o p e at e a c h 1 0 0 p m i n t e r v a l a n d d r a w n o n a r e p l i c a s k e t c h o f the e n t i r e m u s c l e c ross s e c t i o n . T h e l e n g t h o f e a c h s p i n d l e was a p p r o x i m a t e d b y c o u n t i n g the n u m b e r o f c r o s s - s e c t i o n s i n w h i c h the i n t r a f u s a l f i b e r s w e r e seen . T h e n u m b e r o f s p i n d l e s a n d i n t r a f u s a l f i b e r s p e r s p i n d l e a n d the l o c a t i o n o f e a c h r e c e p t o r r e l a t i v e to m y o t e n d o n a n d n e r v e t issue w e r e n o t e d f o r d i a g r a m m a t i c l o n g i t u d i n a l r e c o n s t r u c t i o n s o f s p i n d l e d i s t r i b u t i o n . T h e m u s c l e s p i n d l e i n d e x ( n u m b e r o f m u s c l e s p i n d l e s p e r g r a m m u s c l e w e i g h t ) was c a l c u l a t e d f o r e a c h m u s c l e e x a m i n e d . E l e c t r o n M i c r o s c o p y M i c e at pos tna ta l ages 2 a n d 3 w e e k s w e r e a n e s t h e t i z e d i n t r a p e r i t o n e a l l y w i t h aqueous s o d i u m p e n t o b a r b i t a l at a dosage o f l m g / g m b o d y w e i g h t , a n d p e r f u s i o n was f i x e d w i t h 2.5% g l u t a r a l d e h y d e i n 0.1 M So rensen ' s p h o s p h a t e b u f f e r ( p H 7.3) at r o o m t e m p e r a t u r e . A n i m a l s 1 w e e k o l d w e r e d e c a p i t a t e d , a n d h i n d l e g s w e r e r e m o v e d a n d r a p i d l y i m m e r s e d i n f i x a t i v e . T h e r i g h t E D L a n d so leus w e r e d i s s e c t e d f r o m e a c h a n i m a l a n d m i n c e d w i t h a r a z o r b l ade i n t o 1 to 2 m m 2 segmen ts . M u s c l e p ieces w e r e p l a c e d i n f r e s h f i x a t i v e f o r a f u r t h e r 2 h rs a n d t h e n p o s t f i x e d w i t h 1% a q u e o u s O s 0 4 , d e h y d r a t e d i n g r a d e d e t h a n o l a n d p r o p y l e n e o x i d e , a n d e m b e d d e d i n a n A r a l d i t e / E p o n m i x t u r e . T h i c k sec t i ons ( 0 . 5 - 1 . 0 p m ) o f r a n d o m b l o c k s f r o m e a c h g r o u p w e r e c u t w i t h g lass k n i v e s a n d s t a i n e d w i t h t o l u i d i n e b l u e . S e l e c t e d areas o f the b l o c k s w e r e r e t r i m m e d , a n d u l t r a t h i n sec t i ons c o l l e c t e d o n c o p p e r g r i d s , s t a i n e d w i t h u r a n y l 13 acetate a n d l e a d c i t r a te a n d e x a m i n e d w i t h a P h i l l i p s E M 200 t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e . 14 O B S E R V A T I O N S A to ta l o f 598 m u s c l e s p i n d l e s f r o m the n o r m a l a n d d y s t r o p h i c E D L a n d so leus m u s c l e s w e r e e x a m i n e d i n s e r i a l t ransverse s e c t i o n s , f r o m p r o x i m a l to d i s t a l ends o f e a c h m u s c l e . M o s t s p i n d l e s c o n s i s t e d o f t w o b a g f i b e r s a n d u s u a l l y t w o c h a i n f i b e r s . A s i n o t h e r spec ies (see r e v i e w b y B o y d a n d S m i t h , 1984) , the b a g f i b e r s e x t e n d e d o u t s i d e o f the s p i n d l e c a p s u l e a n d b e y o n d the t e r m i n a t i o n o f the c h a i n f i b e r s . H i s t o c h e m i s t r y I n t r a f u s a l f i b e r s o f the so leus s t a i n e d s i m i l a r l y to those i n the E D L f o r m y o s i n A T P a s e ( F i g s . 1,2) a n d c o u l d be d i f f e r e n t i a t e d i n t o th ree t ypes i n a l l m u s c l e s p i n d l e s . U n d e r a c i d p r e i n c u b a t i o n c o n d i t i o n s , c h a i n f i b e r s w e r e l i g h t l y s t a i n e d , b a g t f i b e r s w e r e u s u a l l y m o d e r a t e l y s t a i n e d , a n d b a g 2 f i b e r s was d a r k l y s t a i n e d . T h i s h i s t o c h e m i c a l p r o f i l e o f i n t r a f u s a l f i b e r s c o u l d be seen b y 1 w e e k o f age i n b o t h the so leus a n d E D L ( F i g s . 1,2), rega rd less o f g e n o t y p e . A t 1 w e e k o f age , b a g 2 f i b e r s a p p e a r e d d a r k l y s t a i n e d a n d c h a i n f i b e r s w e r e l i g h t l y s t a i n e d . T h e s t a i n i n g p a t t e r n o f b a g j f i b e r s w e r e v a r i a b l e . W i t h i n the same zone o f a s p i n d l e , the b a g x f i b e r was o b s e r v e d to be e i t h e r l i g h t l y o r m o d e r a t e l y s t a i n e d , o r s o m e t i m e s as i n t e n s e l y s t a i ned as the b a g 2 f i b e r ( F i g s . 1,2). T h i s s t a i n i n g p r o f i l e was n o t e d i n m u s c l e s p i n d l e p o l a r r eg ions o f b o t h n o r m a l a n d d y s t r o p h i c m u s c l e s . N o changes i n i n t r a f u s a l f i b e r t y p i n g w e r e seen b e t w e e n 1, 2 , a n d 3 w e e k s o f age. H o w e v e r , b y 2 w e e k s b a g x f i b e r s no l o n g e r s t a i n e d as d a r k l y as b a g 2 f i b e r s . S e r i a l sec t i ons t rea ted a f t e r a l k a l i n e p r e i n c u b a t i o n w e r e c o m p a r e d to those a f t e r a c i d p r e i n c u b a t i o n . U n d e r a l k a l i n e c o n d i t i o n s , c h a i n f i b e r s w e r e d a r k l y s t a i ned as w e r e b a g 2 f i b e r s , o n l y the b a g x f i b e r was l i g h t l y s t a i n e d . T h e r e f o r e , b a g 2 f i b e r s w e r e s tab le u n d e r b o t h p H c o n d i t i o n s , w h e r e a s the b a g x s h o w e d o n l y m o d e r a t e s t a b i l i t y a f t e r a c i d p r e i n c u b a t i o n . T h e c h a i n f i b e r s u n d e r w e n t a c o m p l e t e r e v e r s a l i n s t a i n i n g f r o m l i g h t at a c i d p H to d a r k at a l k a l i n e p H . T h u s the s t a i n i n g p a t t e r n e x h i b i t e d b y 15 Figures 1,2 Transverse frozen sections of portions of a normal soleus (Fig. 1) and EDL (Fig. 2) muscles at 1 week of age, stained for myosin ATPase, pH 4.2. Intrafusal fibers of spindles in the soleus appear histochemically similar to those in the EDL. Bag2 (asterisks) and bagj (arrowheads) fibers are more reactive than chain fibers (arrows). Neighbouring extrafusal fibers of types 1 (1) and 2 (2) show differences in staining from each other. Bar=20 um; x 1,200. 16  the c h a i n f i b e r s r e s e m b l e d the t y p i c a l r e v e r s a l o b s e r v e d i n the s u r r o u n d i n g e x t r a f u s a l f i b e r s o f the t y p e 2 v a r i e t y ( F i g s . 3 - 6 ) . I n con t ras t , the b a g f i b e r r e a c t i v i t y d i f f e r e d f r o m that o f the t y p e 2 o r t y p e 1 e x t r a f u s a l f i b e r s ( F i g s . 5 - 6 ) . R e g a r d l e s s o f m u r i n e g e n o t y p e , a n i m a l age , o r m u s c l e p h e n o t y p e , th is p a t t e r n r e m a i n e d cons i s t en t ( F i g s 7 -10) . M y o s i n A T P a s e r e a c t i v i t y o f the th ree i n t r a f u s a l f i b e r t ypes v a r i e d s l i g h t l y a l o n g the l e n g t h o f a n i n d i v i d u a l s p i n d l e . T h e s e v a r i a t i o n s are s u m m a r i z e d s c h e m a t i c a l l y i n F i g u r e 11 . T h e b a g 2 f i b e r s t a i n e d d a r k l y i n the e x t r a c a p s u l a r p o l a r r e g i o n a f t e r a c i d p r e i n c u b a t i o n , a n d g r a d u a l l y lost th is r e a c t i v i t y t o w a r d the e q u a t o r i a l z o n e . U n d e r a l k a l i n e c o n d i t i o n s th is f i b e r s t a i n e d l i g h t l y t h r o u g h o u t i ts l e n g t h . T h e b a g 2 f i b e r s h o w e d a d u a l r e a c t i v i t y u n d e r b o t h p H c o n d i t i o n s , w i t h no r e g i o n a l v a r i a t i o n s . T h e c h a i n f i b e r s w e r e a c i d - l a b i l e a n d a l k a l i n e - s t a b l e , a lso w i t h no r e g i o n a l v a r i a t i o n . T h i s o v e r a l l s t a i n i n g p a t t e r n was t y p i c a l o f n e o n a t a l i n t r a f u s a l f i b e r s o f b o t h the so leus a n d E D L at e a c h age g r o u p s t u d i e d , a n d i t d i d no t d i f f e r b e t w e e n n o r m a l a n d d y s t r o p h i c a n i m a l s . M o r p h o l o g y a n d U l t r a s t r u c t u r e T h e f i r s t o b v i o u s m i c r o s c o p i c changes i n the m u s c l e s e x a m i n e d w e r e d e t e c t e d i n the so leus o f d y s t r o p h i c a n i m a l s at 3 w e e k s o f age . T h e E D L , h o w e v e r , a p p e a r e d u n a f f e c t e d b y d y s t r o p h y i n a l l neona ta l ages s t u d i e d . I n t e r e s t i n g l y , the a l t e ra t i ons i n the so leus o c c u r r e d i n areas o f the m u s c l e s that w e r e r i c h i n m u s c l e s p i n d l e s ( F i g . 12). W i t h i n these areas o f d y s t r o p h i c m u s c l e , the e n d o m y s i u m s u r r o u n d i n g the e x t r a f u s a l f i b e r s as w e l l as m u s c l e s p i n d l e s was i n f i l t r a t e d w i t h a n i n c r e a s e d p o p u l a t i o n o f c o n n e c t i v e t issue ce l l s ( F i g . 12). S o m e o f the e x t r a f u s a l f i b e r s a p p e a r e d to be u n d e r g o i n g p a t h o l o g i c a l changes t y p i c a l o f d y s t r o p h i c m u s c l e . T h e v a r i a b i l i t y i n f i b e r s i ze a n d the p resence o f e x t r a f u s a l f i b e r s w i t h c e n t r a l l y l o c a t e d n u c l e i was g rea te r i n the d y s t r o p h i c m u s c l e s t han i n t h e i r n o r m a l c o u n t e r p a r t s . D e s p i t e these c h a n g e s i n the s u r r o u n d i n g m u s c l e , s p i n d l e s a n d t h e i r i n t r a f u s a l f i b e r s i n d y s t r o p h i c 18 F i g u r e s 3 - 6 S e r i a l sec t i ons o f pa r t o f a n o r m a l ( F i g s . 3,4) a n d d y s t r o p h i c ( F i g s . 5,6) so leus at 3 w e e k s o f age , s t a i n e d f o r m y o s i n A T P a s e a f t e r a c i d ( le f t ) a n d a l k a l i n e ( r i gh t ) p r e i n c u b a t i o n s . I n b o t h n o r m a l a n d d y s t r o p h i c m u s c l e s , p o l a r r eg ions o f s p i n d l e s c o n t a i n bag1 f i b e r s ( a r r o w h e a d s ) that s ta i n m o d e r a t e l y at a c i d p H a n d l i g h t l y at a l k a l i n e p H . B a g 2 f i b e r s (as ter isks) reac t i n t e n s e l y u n d e r b o t h c o n d i t i o n s , w h e r e a s c h a i n f i b e r s ( a r rows ) u n d e r g o a r e v e r s a l f r o m l i g h t i n a c i d to d a r k i n a l k a l i n e . A t y p i c a l r e v e r s a l i n s t a i n i n g is a lso n o t e d i n e x t r a f u s a l f i b e r s o f t ypes 1 (1) a n d 2 (2). B a r = 2 0 p m ; x 1,200. 19 20 F i g u r e s 7 - 1 0 A l t e r n a t e sec t i ons o f pa r t o f a n o r m a l ( F i g s . 7,8) a n d a d y s t r o p h i c ( F i g s . 9 ,10) E D L at 3 w e e k s o f age , s t a i n e d f o r m y o s i n A T P a s e at p H 4.2 ( le f t ) a n d p H 9.4 ( r i gh t ) . B a g 2 f i b e r s (as te r i sks ) a re h i g h l y r e a c t i v e at b o t h p r e i n c u b a t i o n s , w h e r e a s b a g x f i b e r s ( a r r o w h e a d s ) v a r y i n i n t e n s i t y o f s t a i n i n g . C h a i n f i b e r s ( a r rows ) s t a i n s i m i l a r l y to e x t r a f u s a l t y p e 2 (2) f i b e r s , b e i n g a c i d - l a b i l e a n d a l k a l i n e - s t a b l e . B a r = 2 0 p m ; x 1,200. 21 21 F i g u r e 11 S c h e m a t i c d i a g r a m i l l u s t r a t i n g the o v e r a l l m y o s i n A T P a s e s t a i n i n g p r o p e r t i e s o f the b a g ! ( B 1 ) , b a g 2 ( B 2 ) a n d c h a i n ( C ) i n t r a f u s a l f i b e r s i n the n e o n a t a l m o u s e . C o m p o s i t e l o n g i t u d i n a l r e c o n s t r u c t i o n s (above ) w e r e m a d e f r o m t ransve rse s e r i a l sec t i ons ( b e l o w ) . S p i n d l e s w e r e t r a c e d f r o m t h e i r e x t r a c a p s u l a r p o l a r r e g i o n s t h r o u g h to the e q u a t o r i a l z o n e . T h i s is a s y m m e t r i c a l r e p r e s e n t a t i o n o f one h a l f o f a s p i n d l e , c o m p a r i n g i n t r a f u s a l f i b e r r eac t i ons a f t e r a c i d ( le f t ) a n d a l k a l i n e ( r i gh t ) p r e i n c u b a t i o n s . 23  a n i m a l s w e r e s i m i l a r i n a p p e a r a n c e to those o f n o r m a l c o n t r o l s ( F i g . 12). U l t r a s t r u c t u r a l e x a m i n a t i o n was c o n f i n e d to the i n t r a c a p s u l a r r eg ions o f s p i n d l e s , w h e r e b a g a n d c h a i n f i b e r s w e r e eas i l y d i s t i n g u i s h e d ( F i g s . 13 ,14) . A t least t w o s p i n d l e s f r o m e a c h age g r o u p w e r e e x a m i n e d u n d e r the e l e c t r o n m i c r o s c o p e . In e a c h case , the u l t r a s t r u c t u r a l m o r p h o l o g y o f s p i n d l e s i n the d y s t r o p h i c a n i m a l s was s i m i l a r to that i n m u s c l e s o f t he i r n o r m a l c o u n t e r p a r t s . F i g u r e s 13 a n d 14 are e l e c t r o n m i c r o g r a p h s o f a m u s c l e s p i n d l e f r o m a 3 - w e e k d y s t r o p h i c so leus a n d E D L , r e s p e c t i v e l y , a n d rep resen t the t y p i c a l a p p e a r a n c e o f b o t h n o r m a l a n d d y s t r o p h i c s p i n d l e s at th is age . W i t h i n the c e n t r a l e q u a t o r i a l r e g i o n , e a c h s p i n d l e was e n v e l o p e d b y a m u l t i l a y e r e d c a p s u l e c o n s i s t i n g o f c o n c e n t r i c l a m e l l a e o f p e r i n e u r i a l e p i t h e l i a l ce l l s ( F i g . 14). A s i n g l e - l a y e r e d i n n e r c a p s u l e was s u r r o u n d e d b y a c l e a r p e r i a x i a l s p a c e , f r o m w h i c h i t i s o l a t e d the i n t r a f u s a l f i b e r s . L i k e the p e r i n e u r i a l ce l l s o f the o u t e r c a p s u l e , the t h i n i n n e r c a p s u l a r was a lso c o m p o s e d o f f l a t t e n e d , c o n t i g u o u s ce l l s . T h e i n n e r c a p s u l e f o r m e d separa te c o m p a r t m e n t s a r o u n d the i n t r a f u s a l f i b e r s a n d t h e i r s e n s o r y n e r v e e n d i n g s ( F i g . 14). T h e m y o f i l a m e n t c o n t e n t o f b o t h b a g a n d c h a i n f i b e r s was r e d u c e d i n the e q u a t o r i a l z o n e ( F i g . 14). M y o f i l a m e n t s i n the j u x t a e q u a t o r i a l p o r t i o n s o f the s p i n d l e s f o r m e d d i s c r e t e m y o f i b r i l s , o f t e n sepa ra ted b y m i t o c h o n d r i a o f v a r y i n g s izes ( F i g . 13). Sa te l l i t e ce l l s w e r e f o u n d b e n e a t h the e x t e r n a l l a m i n a assoc ia ted w i t h e i t h e r b a g o r c h a i n f i b e r s ( F i g . 13). S e n s o r y t e r m i n a l s a b u n d a n t w i t h m i t o c h o n d r i a w e r e e i t he r s i t ua ted i n s h a l l o w c a v i t i e s o n the i n t r a f u s a l f i b e r s u r f a c e ( F i g . 14) o r p a r t i a l l y e n c i r c l e d the m u s c l e c e l l ( F i g . 13). Q u a n t i t a t i o n a n d D i s t r i b u t i o n T h e to ta l n u m b e r o f m u s c l e s p i n d l e s pe r m u s c l e was t a b u l a t e d f o r e a c h age g r o u p ( T a b l e I). N o d r a m a t i c d i f f e r e n c e s w e r e n o t e d i n m e a n s p i n d l e n u m b e r s b e t w e e n the s l o w a n d fas t m u s c l e s o f e i t he r the n o r m a l o r d y s t r o p h i c a n i m a l s . F o r a m o r e a c c u r a t e assessment o f s p i n d l e d e n s i t y i n the t w o m u s c l e s , the m u s c l e - s p i n d l e i n d e x 25 F i g u r e 12 L i g h t m i c r o g r a p h o f a t ransve rse p l as t i c s e c t i o n o f p o r t i o n o f a 3 - w e e k d y s t r o p h i c so leus , s t a i n e d w i t h t o l u i d i n e b l u e . T h e p o l a r s p i n d l e i n the c e n t e r o f the f i e l d ( a r r o w ) l i es c lose to a n e u r o v a s c u l a r t r u n k a n d appea rs n o r m a l . A n e i g h b o u r i n g a rea o f m u s c l e (as te r i sk ) c o n t a i n s a p l e t h o r a o f d e g e n e r a t i n g a n d a b n o r m a l e x t r a f u s a l f i b e r s a n d c o n n e c t i v e t issue c e l l i n f i l t r a t i o n . Ba r=20 p m ; x 1,100. 26 27 F i g u r e 13 E l e c t r o n m i c r o g r a p h o f a p o r t i o n o f a j u x t a e q u a t o r i a l s p i n d l e f r o m a 3 - w e e k d y s t r o p h i c so leus . Pa r t s o f a b a g f i b e r , a c h a i n f i b e r , a n d a sa te l l i t e c e l l (Sat) are i n d i c a t e d . O u t e r ( O C ) a n d i n n e r ( I C ) c a p s u l a r c o m p o n e n t s a n d a s e n s o r y n e r v e t e r m i n a l ( a r r o w ) a p p e a r n o r m a l . T h e p e r i a x i a l space (as te r i sk ) is c l e a r . B a r = l p m . x l 3 , 6 0 0 . F i g u r e 14 E l e c t r o n m i c r o g r a p h o f pa r t o f a n e q u a t o r i a l s p i n d l e f r o m a 3 - w e e k d y s t r o p h i c E D L . I n n e r c a p s u l e ce l l s a n d t h e i r p rocesses ( I C ) e n v e l o p t w o p a i r s o f b a g (B ) a n d c h a i n ( C ) f i b e r s a n d t h e i r s e n s o r y t e r m i n a l s ( a r r o w ) . A c l e a r p e r i a x i a l space (as te r i sk ) is s u r r o u n d e d b y a n i n t a c t o u t e r c a p s u l e ( O C ) . Ba r=10 u m ; x 4 , 0 0 0 . 28  was a lso c a l c u l a t e d f o r e a c h age g r o u p . T h e m u s c l e - s p i n d l e i n d e x is d e f i n e d as the to ta l n u m b e r o f s p i n d l e s i n a g i v e n m u s c l e e x p r e s s e d p e r m i l l i g r a m o f m u s c l e w e i g h t . Whereas m e a n m u s c l e w e i g h t s i n c r e a s e d s t e a d i l y w i t h a g e , r e f l e c t i n g g r o w t h o f the a n i m a l s , m e a n s p i n d l e n u m b e r s f r o m 1 to 3 w e e k s r e m a i n e d cons tan t f o r e a c h m u s c l e g r o u p . T h i s w o u l d e x p l a i n the g r a d u a l dec rease i n m e a n m u s c l e - s p i n d l e i n d e x f r o m 1 to 3 w e e k s f o r a l l g r o u p s ( T a b l e I). N o o b v i o u s d i f f e r e n c e s w e r e a p p a r e n t i n th is p a r a m e t e r e i t h e r b e t w e e n n o r m a l a n d d y s t r o p h i c a n i m a l s o r b e t w e e n the so leus a n d E D L f o r a g i v e n g r o u p . T h e d i s t r i b u t i o n o f m u s c l e s p i n d l e s i n the so leus was c o m p a r e d to that i n the E D L f o r b o t h n o r m a l a n d d y s t r o p h i c m i c e . L o n g i t u d i n a l r e c o n s t r u c t i o n s w e r e m a d e f r o m s e r i a l t r ansve rse sec t i ons e x t e n d i n g f r o m o r i g i n to i n s e r t i o n ( F i g s . 15 a n d 16). P r i o r to s e c t i o n i n g , the m u s c l e s w e r e m o u n t e d i n s u c h a w a y that m e d i a l a n d la te ra l s ides a n d p o s i t i o n s o f the t e n d o n a n d the e n t r y p o i n t o f the n e u r o v a s c u l a r b u n d l e c o u l d be d e t e r m i n e d . M u s c l e s p i n d l e l eng ths w e r e e s t i m a t e d f r o m the n u m b e r o f se r i a l sec t i ons i n w h i c h the s p i n d l e c o u l d be i d e n t i f i e d . L o n g i t u d i n a l r e c o n s t r u c t i o n s w e r e m a d e f o r a n E D L a n d so leus m u s c l e f r o m e a c h g r o u p s t u d i e d i n b o t h n o r m a l a n d d y s t r o p h i c a n i m a l s . F i g u r e 15 d e p i c t s the d i s t r i b u t i o n o f s p i n d l e s i n a n o r m a l so leus a n d E D L m u s c l e . T h i s t y p i c a l p a t t e r n o f l o c a l i z a t i o n was a lso o b s e r v e d i n m u s c l e s o f d y s t r o p h i c a n i m a l s regard less o f age o r m u s c l e t y p e ( F i g u r e 16). T h e range i n abso lu te n u m b e r o f s p i n d l e s i n the so leus was 9 - 1 4 a n d that i n the E D L 9 - 1 2 . T y p i c a l l y , the s p i n d l e s i n b o t h m u s c l e s w e r e c o n c e n t r a t e d a l o n g the n e u r o v a s c u l a r p a t h w a y . 30 T A B L E I. C o m p a r i s o n o f m e a n s p i n d l e n u m b e r a n d m e a n s p i n d l e i n d e x f o r n o r m a l ( N ) a n d d y s t r o p h i c ( D Y ) so leus a n d E D L m u s c l e s at 1 , 2 , a n d 3 w e e k s o f age . 1 W K 2 W K 3 W K N o . I n d e x N o . I n d e x N o . I n d e x S O L E U S N 9.5 9.0 12 .0* 4.2 11.3 2.8 D Y 10.4 8.2 10.0 3.3 10.4 2.9 E D L N 10.5 8.0 11.2 3.8 1 1 . 3 * 2.5 D Y 10.4 7.1 9.2 2.9 9.2 2.4 * P < 0.05 b e t w e e n N a n d D Y g r o u p s , as d e t e r m i n e d b y S t u d e n t ' s t - tes t . C o m p a r i s o n s w e r e a lso m a d e b e t w e e n the so leus a n d E D L across a l l g r o u p s , b u t these d i d no t p r o v e s i g n i f i c a n t at p<0.05. n=5, w i t h the e x c e p t i o n o f the 1 - w e e k n o r m a l g r o u p s a n d the 2 - w e e k n o r m a l E D L s p i n d l e i n d e x d a t a , w h e r e n=4. 31 F i g u r e 15 L o n g i t u d i n a l r e c o n s t r u c t i o n s ( F i g . 15a) s h o w i n g the d i s t r i b u t i o n o f m u s c l e s p i n d l e s i n the so leus ( S O L ) a n d E D L m u s c l e s at 3 w e e k s o f age . E a c h l i n e i n the d i a g r a m des igna tes a s p i n d l e . O r i g i n ( O ) , i n s e r t i o n (I) a n d m e d i a l ( M ) a n d l a te ra l ( L ) b o r d e r s o f e a c h m u s c l e a re i n d i c a t e d . F i g u r e 15b d e p i c t s the c o r r e s p o n d i n g t ransverse r e c o n s t r u c t i o n s o f the n o r m a l so leus a n d E D L s h o w i n g the d i s t r i b u t i o n o f m u s c l e s p i n d l e s f r o m o r i g i n to i n s e r t i o n . E a c h do t rep resen ts a m u s c l e s p i n d l e . D o r s a l ( D ) a n d v e n t r a l ( V ) s u r f a c e s are i n d i c a t e d as w e l l as the m e d i a l ( M ) a n d l a te ra l ( L ) b o r d e r s . T h e i n t e r v a l b e t w e e n e a c h t ransve rse s e c t i o n is 1000 u m . 32  F i g u r e 16 L o n g i t u d i n a l r e c o n s t r u c t i o n s ( F i g . 16a) s h o w i n g the d i s t r i b u t i o n o f m u s c l e s p i n d l e s i n the d y s t r o p h i c so leus ( S O L ) a n d E D L m u s c l e s at 3 w e e k s o f age . A s i n F i g u r e 15 , e a c h l i n e rep resen ts a m u s c l e s p i n d l e as t hey w e r e o b s e r v e d f r o m the o r i g i n (O ) to i n s e r t i o n (I). F i g u r e 16b shows the c o r r e s p o n d i n g d i s t r i b u t i o n i n t ransverse r e c o n s t r u c t i o n s . E a c h do t i n d i c a t e s a m u s c l e s p i n d l e a n d i n t e r v a l s b e t w e e n t r a c e d t ransverse sec t i ons are 1000 u m . M e d i a l ( M ) , l a t e ra l ( L ) , d o r s a l ( D ) a n d v e n t r a l b o r d e r s a re i n d i c a t e d . 34 © H \0OfU ® SOL EDL D I S C U S S I O N T h e resu l ts o f the p resen t s t u d y c o m p l e m e n t p r e v i o u s o b s e r v a t i o n s that m u s c l e s p i n d l e s i n d y s t r o p h i c m u r i n e m u s c l e s are m o r p h o l o g i c a l l y u n a f f e c t e d b y the d isease p rocess ( M e i e r , 1969; Y e l l i n , 1974b; J a m e s a n d M e e k , 1979) . In a d d i t i o n , w e have s h o w n tha t n e o n a t a l s p i n d l e s i n the d y s t r o p h i c s l o w - t w i t c h so leus a n d f a s t - t w i t c h E D L m u s c l e s r e s e m b l e those f r o m t h e i r a g e - m a t c h e d n o r m a l c o n t r o l s . O u r resu l ts c o n f i r m those p r e v i o u s l y r e p o r t e d ( M e i e r , 1969; Y e l l i n , 1974b) bu t a lso i n d i c a t e tha t , i n con t ras t to the changes i n e x t r a f u s a l f i b e r s , i n t r a f u s a l f i b e r s i n d y s t r o p h i c m u s c l e s d i f f e r e n t i a t e n o r m a l l y i n to th ree t ypes . In b o t h n o r m a l a n d d y s t r o p h i c a n i m a l s , b a g x , b a g 2 a n d c h a i n f i b e r s c o u l d be d i f f e r e n t i a t e d o n the bas is o f A T P a s e r e a c t i v i t y a f t e r a c i d a n d a l k a l i n e p r e i n c u b a t i o n s . R e g i o n a l v a r i a t i o n s i n s t a i n i n g c h a r a c t e r i s t i c s s i m i l a r to those n o t e d i n o t h e r m u s c l e s a n d s p e c i e s , n a m e l y the rat ( S o u k u p , 1976; B a n k s et a l . , 1977; K h a n a n d S o u k u p , 1979) , ca t ( B a n k s et a l . , 1977; B a k k e r a n d R i c h m o n d , 1981 ; K u c e r a , 1981) , m o n k e y ( O v a l l e a n d S m i t h , 1972) , a n d h u m a n ( K u c e r a a n d D o r o v i n i - z i s , 1979) w e r e a lso o b s e r v e d i n the m o u s e . W e h a v e s h o w n tha t , as i n o t h e r s p e c i e s , b a g 2 f i b e r s i n the m o u s e are a c i d - s tab le a n d h a v e l o w a l k a l i a c t i v i t y . B a g 2 f i b e r s , i n c o n t r a s t , a re a c i d - s t a b l e a n d d i s p l a y h i g h a l k a l i r e a c t i v i t y , w h e r e a s c h a i n f i b e r s a re a c i d - l a b i l e w i t h h i g h a l k a l i a c t i v i t y . T h e s e s t a i n i n g pa t te rns w e r e c o n s i s t e n t l y o b s e r v e d i n e x t r a c a p s u l a r p o l a r r e g i o n s o f m u s c l e s p i n d l e s . In con t ras t to p r e v i o u s r e p o r t s , h o w e v e r , s l i gh t r e g i o n a l d i f f e r e n c e s w e r e o b s e r v e d i n the m o u s e w h e n c o m p a r e d to o t h e r s p e c i e s . W h e r e a s o u r resu l t s s h o w that b a g t f i b e r s e x h i b i t l o w a l k a l i a c t i v i t y o v e r the e n t i r e l e n g t h o f the s p i n d l e , K u c e r a (1977) has r e p o r t e d that the deg ree o f a l k a l i s t a b i l i t y v a r i e s f r o m c a p s u l a r to p o l a r r e g i o n s . B a n k s a n d c o w o r k e r s (1977) n o t e d the bag1 f i b e r s i n r a b b i t , ra t a n d ca t s p i n d l e s are m o r e r e a c t i v e i n e x t r a c a p s u l a r p o l a r r e g i o n s u n d e r a l k a l i n e c o n d i t i o n s , a f e a t u r e s i m i l a r to the b a g 2 f i b e r s . O n the o t h e r h a n d , u n d e r a c i d c o n d i t i o n s , the r e g i o n a l v a r i a t i o n s n o t e d b y us i n b a g x f i b e r s o f the m o u s e are 36 s i m i l a r to those r e p o r t e d i n o the r spec ies . T h u s , as s e r i a l sec t i ons o f a s p i n d l e are f o l l o w e d f r o m the p o l a r e n d t o w a r d the e q u a t o r , the s t a i n i n g p a t t e r n g r a d u a l l y changes i n a s e q u e n t i a l f a s h i o n f r o m d a r k to l i gh t . It has b e e n sugges ted that v a r i a t i o n s i n m y o s i n A T P a s e s t a i n i n g o f the i n t r a f u s a l f i b e r s m a y be d e p e n d e n t o n m o t o r i n n e r v a t i o n ( Y e l l i n , 1969; M i l b u r n , 1973) . T h i s h y p o t h e s i s , h o w e v e r , has b e e n d i s p u t e d b y Z e l e n a a n d S o u k u p (1974) w h o s h o w e d that rat s p i n d l e s s e l e c t i v e l y d e - e f f e r e n t e d at b i r t h e v e n t u a l l y d i f f e r e n t i a t e a n d m a t u r e to o b t a i n t h e i r n o r m a l h i s t o e n z y m a t i c c h a r a c t e r i s t i c s . A s w e l l , K u c e r a (1981) f o u n d no c o r r e l a t i o n b e t w e e n r e g i o n a l A T P a s e a c t i v i t y a n d the l o c a t i o n , n u m b e r , o r t y p e o f m o t o r e n d i n g s d e t e r m i n e d b y cho l i nes te rase s t a i n i n g . H e sugges ted that the h i s t o c h e m i c a l response o f i n t r a f u s a l f i b e r s m a y no t be g o v e r n e d b y the same fac to r s that c o n t r o l e x t r a f u s a l f i b e r t y p i n g . T h e i n t e g r i t y o f the i n t r a f u s a l f i b e r s m a y i ns tead be i n f l u e n c e d b y be ta i n n e r v a t i o n i n p o l a r r e g i o n s , o r b y s e n s o r y i n n e r v a t i o n i n the c o n f i n e d e n v i r o n m e n t o f the c a p s u l e . It is a lso p o s s i b l e that d i f f e r e n t r eg ions o f the i n t r a f u s a l f i b e r s i n a n i n d i v i d u a l s p i n d l e m a y c o n t a i n d i f f e r e n t m y o s i n i s o f o r m s . I m m u n o h i s t o c h e m i c a l r epo r t s ( P i e r o b o n B o r m i o l i , 1980; C e l i o , 1981 ; et K r o n n i e , 1981 ,1982) h a v e r e c e n t l y p r e s e n t e d e v i d e n c e that there are th ree m y o s i n t ypes assoc ia ted w i t h i n t r a f u s a l f i b e r s . B a g 2 f i b e r s reac t to a n t i - s l o w m y o s i n , c h a i n f i b e r s to a n t i - f a s t m y o s i n , a n d b a g x f i b e r s to b o t h a n t i - s l o w a n d a n t i - s l o w t o n i c m y o s i n . F u t u r e s tud ies d e s i g n e d to e v a l u a t e s e r i a l sec t i ons o f a n e n t i r e s p i n d l e s t a i n e d w i t h v a r i o u s a n t i m y o s i n a n t i b o d i e s m a y r e v e a l r e g i o n a l v a r i a t i o n s i n m y o s i n i s o f o r m s as w e l l . It is n o t e w o r t h y that m u s c u l a r d y s t r o p h y i n the m o u s e c a n d r a m a t i c a l l y a f f e c t e x t r a f u s a l f i b e r t y p i n g as e a r l y as th ree w e e k s o f age ( P a r r y a n d P a r s l o w , 1981 ; W i r t z et a l . , 1983) P e r h a p s the pos tna ta l age range se lec ted i n th is s t u d y was too e a r l y to de tec t a l t e ra t i ons i n i n t r a f u s a l f i b e r t y p e i n response to the d i sease . H i s t o c h e m i c a l changes i n ra t e x t r a f u s a l f i b e r s i n response to d e n e r v a t i o n o r e x e r c i s e are p r e c e d e d b y t r a n s i t i o n s i n o t h e r p r o t e i n s s u c h as p a r v a l b u m i n a n d the p e p t i d e p a t t e r n o f 37 s a r c o p l a s m i c r e t i c u l u m ( G r e e n et a l . , 1984; M u n t e n e r et a l . , 1985) . W h i l e a s i m i l a r p h e n o m e n o n m a y o c c u r i n response to d y s t r o p h y i n the m o u s e , i t is a lso poss ib l e that m y o s i n A T P a s e s t a i n i n g m e t h o d s are no t s e n s i t i v e i n d i c a t o r s o f e a r l y a b n o r m a l f i b e r t ype . I m m u n o h i s t o c h e m i c a l s tud ies w i t h a n t i s e r a to v a r i o u s m y o s i n l i g h t c h a i n s a n d h e a v y c h a i n s m i g h t r e v e a l a m o r e r e l i a b l e m a r k e r f o r a l t e ra t i ons i n f i b e r t y p e . M u s c l e s p i n d l e s m a y be s p a r e d m a n i f e s t a t i o n s o f d isease b y s o m e p r o t e c t i v e na tu re o f the o u t e r a n d i n n e r c a p s u l e s , w h i c h are k n o w n to r egu la te the i n t e r n a l e n v i r o n m e n t o f these recep to r s ( D o w et a l . , 1980) . O n l y r e c e n t l y h a v e a n y s tud ies been c o n d u c t e d to i n v e s t i g a t e the ex ten t to w h i c h the c a p s u l e m a y i n f l u e n c e the c a p a c i t y o f the s p i n d l e to m a i n t a i n n o r m a l f u n c t i o n i n the f a c e o f a n a b n o r m a l e n v i r o n m e n t . It has b e e n s h o w n that s p i n d l e f o r m a t i o n i n r e g e n e r a t i n g m a m m a l i a n m u s c l e f o l l o w i n g m u s c l e g ra f t s d e p e n d s o n the s u r v i v a l o f the c a p s u l e ( R o g e r s a n d C a r l s o n , 1981) . I nadequa te senso ry i n n e r v a t i o n i n the f a c e o f a n i n t a c t a n d v i a b l e c a p s u l e , h o w e v e r , resu l ts i n a b n o r m a l pa t te rns o f m y o s i n A T P a s e a c t i v i t y i n i n t r a f u s a l f i b e r s o f r e g e n e r a t i n g m u s c l e ( R o g e r s , 1982) . W h e n m u s c l e s p i n d l e s are s e l e c t i v e l y d e a f f e r e n t e d , the p e r i a x i a l f l u i d space is s i g n i f i c a n t l y d e c r e a s e d a n d o n l y the i n t r a c a p s u l a r m y o s i n A T P a s e s t a i n i n g p a t t e r n i n b a g f i b e r s is a l t e red ( K u c e r a , 1980). T h i s a b n o r m a l i t y m a y r e f l e c t the loss o f t r o p h i c i n t e r a c t i o n b e t w e e n a f f e r e n t i n n e r v a t i o n a n d the m u s c l e s p i n d l e c a p s u l e , t h e r e b y d i m i n i s h i n g the r e g u l a t o r y f u n c t i o n o f the c a p s u l e . S p i n d l e s i n s l o w - t w i t c h ve rsus f a s t - t w i t c h m u s c l e s T h e m u s c l e s p i n d l e p o p u l a t i o n d e n s i t y is u s u a l l y d e t e r m i n e d b y the hos t m u s c l e ' s f u n c t i o n ( V o s s , 1937; C o o p e r , 1960; B a r k e r , 1974) . G e n e r a l l y , the s p i n d l e d e n s i t y is h i g h e r i n m u s c l e s i n i t i a t i n g f i n e m o v e m e n t s , m a i n t a i n i n g p o s t u r e , o r e x h i b i t i n g s l o w c o n t r a c t i l e p r o p e r t i e s ( B a r k e r a n d C h i n , 1960; Swe t t a n d E l d r e d , 1960; Y e l l i n , 1969) . O u r o b s e r v a t i o n s i n d i c a t e that s p i n d l e s i n the s l o w - t w i t c h so leus d o no t d i f f e r h i s t o c h e m i c a l l y f r o m those i n the f a s t - t w i t c h E D L w i t h respec t to m y o s i n A T P a s e 38 a c t i v i t y . I n a d d i t i o n , the n u m b e r a n d d i s t r i b u t i o n o f s p i n d l e s w e r e no t s i g n i f i c a n t l y d i f f e r e n t b e t w e e n these t w o m u s c l e s i n neona ta l a n i m a l s . O u r h i s t o c h e m i c a l f i n d i n g s c o m p l e m e n t resu l ts r e p o r t e d b y o the rs f o r b a g f i b e r s i n rat so leus a n d E D L musc les ( S o u k u p , 1976 , et K r o n n i e , 1981) . S o u k u p (1976) h o w e v e r , e m p l o y e d the G u t h a n d S a m a h a (1970) s t a i n i n g p r o t o c o l a n d n o t e d that c h a i n f i b e r s i n the so leus r e a c t e d w i t h v a r i a b l e i n tens i t i es a f t e r a c i d p r e i n c u b a t i o n , w h e r e a s the E D L c o n s i s t e n t l y d i s p l a y e d the t y p i c a l l i g h t s t a i n i n g p a t t e r n f o r m y o s i n A T P a s e . T h i s s l i g h t d i f f e r e n c e i n the so leus a n d E D L c h a i n f i b e r r e a c t i v i t i e s was no t o b s e r v e d i n the p resen t s t u d y , n o r has i t b e e n r e p o r t e d b y o the rs . In the p resen t s t u d y , the m e a n n u m b e r o f s p i n d l e s i n b o t h the so leus a n d E D L i n a l l age g r o u p s was a p p r o x i m a t e l y t en . T h i s n u m b e r is s i m i l a r to c o u n t s m a d e b y o the rs f o r the so leus ( Y e l l i n , 1974a) a n d E D L ( K o z e k a a n d O n t e l l , 1981) o f the m o u s e . S l i g h t l y h i g h e r ave rages h a v e b e e n p u b l i s h e d f o r s i m i l a r m u s c l e s i n the rat ( Y e l l i n , 1969; K u c e r a , 1977) . A s a g e n e r a l r u l e , s p i n d l e d e n s i t y is g rea te r i n m u s c l e s that assist i n p o s t u r a l m a i n t e n a n c e , s u c h as the so leus ( B a r k e r , 1974) . O u r resu l ts d i d no t s h o w a s i g n i f i c a n t d i f f e r e n c e i n s p i n d l e d e n s i t y i n the so leus f r o m that o f the E D L i n a n y o f the n e o n a t a l g r o u p s s t u d i e d . A n e x p l a n a t i o n f o r th is o b s e r v a t i o n m a y be that s p i n d l e d e n s i t y is no t a r e l i a b l e m e a s u r e w h e n a p p l i e d to d e v e l o p i n g m u s c l e s i n c e , o w i n g to g r o w t h , the w e i g h t o f the m u s c l e has no t ye t s t a b i l i z e d . A s e x p e c t e d , the d i s t r i b u t i o n o f m u s c l e s p i n d l e s f o l l o w e d the c o u r s e o f the n e u r o v a s c u l a r t r u n k i n b o t h types o f m u s c l e . T h i s p a t t e r n was no t a l t e red b y d y s t r o p h y o r age o f the a n i m a l . O w i n g to the h e t e r o g e n e i t y o f e x t r a f u s a l f i b e r types i n b o t h the so leus a n d the E D L o f the m o u s e , no seg rega t i on o f s p i n d l e s w i t h i n a p a r t i c u l a r a rea o f the m u s c l e was n o t e d . T h i s o b s e r v a t i o n is i n a g r e e m e n t w i t h p r e v i o u s repo r t s o f the d i s t r i b u t i o n o f s p i n d l e s i n se lec ted h i n d l i m b m u s c l e s o f the rat ( Y e l l i n , 1969) , a n d m o u s e ( K o z e k a a n d O n t e l l , 1981) . 39 M o r p h o g e n e s i s M u s c l e s p i n d l e d e v e l o p m e n t i n the m o u s e beg ins at d a y 15 i n u t e r o ( K o z e k a a n d O n t e l l , 1981) , a b o u t 3 d a y s p r i o r to that r e p o r t e d i n the rat ( M i l b u r n , 1973) . T h e d e f i n i t i v e n u m b e r o f m u s c l e s p i n d l e s i n the m o u s e is e s t a b l i s h e d 2 d a y s a f t e r p r i m i t i v e s e n s o r y con tac t s a re m a d e b e t w e e n n e r v e f i b e r s a n d d e v e l o p i n g m y o t u b e s . In a d d i t i o n , the n u m b e r o f i n t r a f u s a l m y o t u b e s i n adu l t s has b e e n s h o w n to be p resen t at b i r t h ( K o z e k a a n d O n t e l l , 1981) . A g a i n , th is is e a r l i e r t h a n s i m i l a r even ts r e p o r t e d f o r m u s c l e s p i n d l e s i n the ra t , w h e r e the f u l l c o m p l e m e n t o f i n t r a f u s a l f i b e r s is e s t a b l i s h e d b y 4 d a y s o f age ( L a n d o n , 1972; M i l b u r n , 1973) . F u r t h e r m o r e , p o l a r r e g i o n s o f these f i b e r s are s t i l l i n a n i m m a t u r e state a n d d o no t o b t a i n d i f f e r e n t i a t i o n o f t h e i r d i s t i n c t a d u l t f i b e r t y p e u n t i l 12 d a y s p o s t n a t a l l y ( M i l b u r n , 1973) . T h e f i n d i n g s o f K o z e k a a n d O n t e l l (1981) s t r o n g l y s u p p o r t the h y p o t h e s i s p r o p o s e d b y L a n d o n (1972) a n d M i l b u r n (1973) that success i ve g e n e r a t i o n s o f n e w i n t r a f u s a l m y o t u b e s o c c u r b y f u s i o n o f p r i m i t i v e m o n o n u c l e a t e d ce l l s . I n a d d i t i o n , the n e w l y f o r m e d m y o t u b e s a p p e a r to o r i g i n a t e i n c lose a f f i l i a t i o n w i t h the s e n s o r y r e g i o n o f the p r i m a r y m y o t u b e ( K o z e k a a n d O n t e l l , 1981) . T h i s c lose r e l a t i o n s h i p has a p r o f o u n d i n f l u e n c e o n d i f f e r e n t i a t i o n o f the i n t r a f u s a l f i b e r t y p e . T h e resu l ts o f the p resen t s t u d y i n d i c a t e that the a d u l t h i s t o c h e m i c a l p r o f i l e o f i n t r a f u s a l f i b e r s is p resen t b y 1 w e e k o f age i n the m o u s e , regard less o f g e n o t y p e . In b o t h the so leus a n d E D L , m y o s i n A T P a s e s t a i n i n g at 1 w e e k was the same as that n o t e d at 2 a n d 3 w e e k s o f age w i t h one e x c e p t i o n . A t 1 w e e k o f age , the b a g x f i b e r f r e q u e n t l y s t a i n e d as i n t e n s e l y as the b a g 2 f i b e r u n d e r a c i d c o n d i t i o n s (see F i g . 1) f o r a d i s t a n c e that e x t e n d e d i n t o the c a p s u l a r z o n e . T h i s was not o b s e r v e d at 2 o r 3 w e e k s . It is p o s s i b l e , t h e r e f o r e , that the h i s t o c h e m i c a l d i f f e r e n t i a t i o n o f the b a g x f i b e r lags b e h i n d that o f the o t h e r i n t r a f u s a l f i b e r s . W h i l e i t has b e e n r e p o r t e d that m a m m a l i a n b a g f i b e r s d e v e l o p i n c lose a s s o c i a t i o n w i t h e a c h o t h e r ( K o z e k a a n d O n t e l l , 1981 ; B a r k e r a n d M i l b u r n , 1984) , i t has a lso b e e n s h o w n that b a g 2 f i b e r s a n d c h a i n f i b e r s share a s i n g l e c o m p a r t m e n t w i t h i n a d e v e l o p i n g s p i n d l e ( B u t l e r , 1979 , 1980). 40 T h u s , i f b a g x f i b e r s i n the m o u s e w e r e to d e v e l o p as a separa te g r o u p , one m i g h t e x p e c t the t i m e c o u r s e o f t h e i r h i s t o c h e m i c a l d i f f e r e n t i a t i o n to v a r y f r o m that o f b a g 2 a n d c h a i n f i b e r s . T h e v a r i a b i l i t y i n d i f f e r e n t i a t i o n i n t o d i s t i n c t i n t r a f u s a l f i b e r t ypes m a y be i n f l u e n c e d b y the g r a d u a l e l o n g a t i o n o f the s p i n d l e c a p s u l e f r o m the e q u a t o r i a l s e n s o r y r e g i o n to the e x p o s e d p o l a r ends ( K o z e k a a n d O n t e l l , 1981) . W h i l e it has b e e n s h o w n tha t s e n s o r y i n n e r v a t i o n is necessa ry f o r s p i n d l e d i f f e r e n t i a t i o n ( Z e l e n a , 1957) a n d tha t f u s i m o t o r i n n e r v a t i o n is r e q u i r e d f o r the d e v e l o p m e n t o f m y o s i n c h a r a c t e r i s t i c s (et K r o n n i e , 1982) , the deg ree o f i n f l u e n c e o f the s p i n d l e c a p s u l e o n d i f f e r e n t i a t i o n o f f i b e r t y p e is u n k n o w n . F u n c t i o n a l a n d c l i n i c a l c o n s i d e r a t i o n s In a g r e e m e n t w i t h the i n i t i a l f i n d i n g s o f J a m e s a n d M e e k (1979 ) , n o n e o f the s p i n d l e s i n the d y 2 J d y s t r o p h i c m u s c l e s a p p e a r e d u l t r a s t r u c t u r a l l y a b n o r m a l at a n y age g r o u p e x a m i n e d i n the p resen t s t u d y . M o r p h o l o g i c a l l y , no s igns o f c a p s u l e t h i c k e n i n g , e n l a r g e m e n t o f the p e r i a x i a l s p a c e , o r i n t r a f u s a l f i b e r a t r o p h y c o u l d be d e t e c t e d . T h e s e o b s e r v a t i o n s a lso c o n f i r m the e a r l i e r l i g h t m i c r o s c o p i c s tud ies o f M e i e r (1969) a n d Y e l l i n ( 1974b ) i n the d y s t r a i n o f d y s t r o p h i c m o u s e . P a t h o l o g i c a l changes s u c h as i n t r a f u s a l f i b e r s p l i t t i n g , c o l l a g e n o u s t h i c k e n i n g o f the c a p s u l e , a n d a b n o r m a l i n n e r v a t i o n are c o m m o n f i n d i n g s i n h u m a n d y s t r o p h i a m y o t o n i a ( S w a s h a n d F o x , 1975; M a y n a r d et a l . , 1977) . W h i l e some a l te ra t ions i n s p i n d l e m o r p h o l o g y h a v e a lso been n o t e d i n t e r m i n a l stages o f m u s c u l a r d y s t r o p h y i n h u m a n s ( S w a s h a n d F o x , 1976) a n d m i c e ( D o w a n d O v a l l e , 1985) , t h e i r f u n c t i o n a l i m p l i c a t i o n s r e m a i n e n i g m a t i c . In h u m a n D u c h e n n e a n d c o n g e n i t a l m u s c u l a r d y s t r o p h y , the ex ten t to w h i c h m u s c l e s p i n d l e s are a f f e c t e d is r e l a ted to the stage o f the d isease p rocess ( C a z z a t o a n d W a l t o n , 1968) . I n p r e c l i n i c a l o r e a r l y d y s t r o p h y , the p r o p o r t i o n o f a f f e c t e d s p i n d l e s is d i m i n i s h e d , a n d the degree o f p a t h o l o g i c a l c h a n g e is less p r o m i n e n t t h a n that seen i n m o r e a d v a n c e d cases. It w o u l d a p p e a r t h e r e f o r e that m u s c l e s p i n d l e s 41 m a y be a f f e c t e d i n the p r o g r e s s i v e c o u r s e o f the d isease a n d tha t a n y changes that m a y o c c u r w i t h i n the s p i n d l e d u r i n g m o r e a d v a n c e d stages o f d y s t r o p h y are p r o b a b l y n o n s p e c i f i c l o n g - t e r m responses that are not d i r e c t l y r e l a ted to the p r i m a r y e t i o l o g y o f the d isease i t se l f . T h e c o m p l e x p o l y n e u r o n a l i n n e r v a t i o n o f m u s c l e s p i n d l e s has b e e n the s u b j e c t o f e x t e n s i v e r e v i e w s ( B a r k e r , 1974; K e n n e d y et a l . , 1980; B o y d a n d S m i t h , 1984). H o w e v e r , o u r k n o w l e d g e o f the e x a c t p a t t e r n a n d f u n c t i o n a l s i g n i f i c a n c e o f the d u a l s e n s o r y a n d f u s i m o t o r i n n e r v a t i o n is s t i l l i n c o m p l e t e . It is t e m p t i n g to specu la te that sub t l e c h a n g e s i n n e u r o n a l a c t i v i t y that a re no t r e f l e c t e d i n s t r u c t u r a l c h a r a c t e r i s t i c s i n the s p i n d l e d o o c c u r i n d y s t r o p h y , a n d that th is f u n c t i o n a l a l t e r a t i o n is r e f l e c t e d i n the r e l a t i v e l y e a r l y onset o f a b n o r m a l t e n d o n r e f l e x e s n o t e d i n h u m a n m u s c u l a r d y s t r o p h y ( W a l t o n a n d G a r d n e r - M e d w i n , 1981) . I f e x t r a f u s a l f i b e r d e g e n e r a t i o n is p a r t i a l l y the resu l t o f a n a b n o r m a l n e u r o t r o p i c i n f l u e n c e (Sa i to et a l . , 1983) , i t is n o t e w o r t h y that a m o r p h o l o g i c a l l y a b n o r m a l n e r v e , s u c h as that o b s e r v e d i n the d y 2 J a l l e l i c s t r a i n o f m o u s e ( B r a y et a l . , 1977; M o n t g o m e r y a n d S w e n a r c h u k , 1978) , does no t a p p e a r to a f f e c t the d e v e l o p m e n t o f m u s c l e s p i n d l e s o r the d i f f e r e n t i a t i o n o f i n t r a f u s a l f i b e r s . It is poss ib l e that the p o l y n e u r o n a l i n n e r v a t i o n , u n i q u e to i n t r a f u s a l f i b e r s i n m a t u r e m u s c l e , p r o v i d e s a d e f e n s e m e c h a n i s m f o r the s p i n d l e d u r i n g i n c i p i e n t stages o f the d i sease . U n f o r t u n a t e l y the d e v e l o p m e n t o f s p i n d l e a b n o r m a l i t i e s i n m u s c u l a r d y s t r o p h y is no t f u l l y u n d e r s t o o d . T h i s m a y be d u e to the f a c t that no t o n l y h a v e f e w s p i n d l e s b e e n s t u d i e d i n th is n e u r o m u s c u l a r d isease bu t t h e y h a v e o n l y r e c e i v e d c u r s o r y i n s p e c t i o n . W i t h the a i d o f m o r e sens i t i ve b i o c h e m i c a l a n d i m m u n o l o g i c a l m a r k e r s w e m a y be t te r u n d e r s t a n d the r o l e o f the m u s c l e s p i n d l e i n n o r m a l m u s c l e a n d its a l t e r a t i o n i n m u s c u l a r d y s t r o p h y . O n e s u c h i m m u n o l o g i c a l m a r k e r c o u l d be the c a l c i u m - b i n d i n g p r o t e i n p a r v a l b u m i n . T h i s p r o t e i n is assoc ia ted w i t h f a s t - t w i t c h m u s c l e f i b e r s a n d the deg ree o f p a r v a l b u m i n i m m u n o r e a c t i v i t y i n m u s c l e f i b e r s has b e e n e s t a b l i s h e d as a n a l t e r n a t i v e to m y o s i n A T P a s e f i b e r t y p i n g ( C e l i o a n d H e i z m a n n , 42 1982) . In a p r e v i o u s r e p o r t f r o m T h e M u s c u l a r D y s t r o p h y G r o u p at the U n i v e r s i t y o f B r i t i s h C o l u m b i a p a r v a l b u m i n was n o t e d to be s i g n i f i c a n t l y a l t e r e d i n b o t h fas t a n d s l o w - t w i t c h m u s c l e s o f a d u l t d y s t r o p h i c m i c e ( J a s c h a n d M o a s e , 1985) . I n a d d i t i o n , changes i n the c a l c i u m - b i n d i n g a n d seques te r i ng s y s t e m o c c u r e a r l i e r t h a n the t r a n s i t i o n o f the i s o f o r m s o f m y o s i n i n response to e l e c t r o - s t i m u l a t i o n (Sre ter et a l . , 1973; H e i l m a n n a n d P e t t e , 1979; K l u g et a l . , 1983b ; G r e e n et a l . , 1984) . T h e r e f o r e , p a r v a l b u m i n , as a n i m m u n o c h e m i c a l m a r k e r , m a y de tec t the e x p r e s s i o n o f d y s t r o p h y i n e x t r a f u s a l a n d i n t r a f u s a l f i b e r s at a n e a r l i e r stage t h a n m y o s i n A T P a s e h i s t o c h e m i s t r y . T h e r e m a i n d e r o f th is thes is w i l l d e a l w i t h the l o c a l i z a t i o n o f p a r v a l b u m i n i n i n t r a f u s a l a n d e x t r a f u s a l f i b e r s a n d q u a n t i f i c a t i o n o f p a r v a l b u m i n i n n o r m a l a n d d y s t r o p h i c m i c e . 43 C H A P T E R 2. B i o c h e m i c a l a n d I m m u n o c h e m i c a l l o c a l i z a t i o n o f p a r v a l b u m i n I N T R O D U C T I O N H i s t o r i c a l B a c k g r o u n d o f P a r v a l b u m i n D e u t i c k e (1934) d e s c r i b e d s l o w s e d i m e n t i n g f r a c t i o n s i n f r o g m u s c l e at l o w sal t c o n c e n t r a t i o n s u s i n g u l t r a c e n t r i f u g a t i o n . H e n r o t t e (1955) s u b s e q u e n t l y o b s e r v e d s i m i l a r l o w m o l e c u l a r w e i g h t , a c i d i c p r o t e i n s w i t h a n u n u s u a l u l t r a v i o l e t a b s o r p t i o n s p e c t r u m f r o m c a r p m u s c l e . P e c h e r e (1968) r e f e r r e d to these p r o t e i n s as p a r v a l b u m i n s , based o n t h e i r s m a l l s i ze a n d h i g h w a t e r s o l u b i l i t y . In l o w e r v e r t e b r a t e s , as m a n y as 6 i so t ypes o f p a r v a l b u m i n ( P V ) h a v e b e e n i so la ted f r o m the same m u s c l e ( F o c a n t a n d P e c h e r e , 1965) , w h e r e a s h i g h e r ve r teb ra tes c o n t a i n a s i ng l e i s o t y p e i n a g i v e n m u s c l e ( P e c h e r e et a l . , 1971 ; H a i e c h et a l . , 1979b) . C a r p m u s c l e , w h i c h c o n t a i n s 4 m a i n i so t ypes o f P V , s o o n b e c a m e the s u b j e c t o f the f i r s t t h r e e - d i m e n s i o n a l c r y s t a l l i z a t i o n d e s c r i p t i o n s ( N o c k o l d s et a l . , 1972) o f a l l the r e l a t ed C a 2 + - b i n d i n g p r o t e i n s ( T r o p o n i n - C , c a l m o d u l i n , m y o s i n l i g h t c h a i n s ) . B a s e d o n p e p t i d e m a p s ( P e c h e r e a n d C a p o n y , 1969) , i t was c o n c l u d e d that these p r o t e i n s cons t i t u te a f a m i l y o f h o m o l o g o u s p r o t e i n s . M o r e i m p o r t a n t l y , P e c h e r e a n d c o w o r k e r s (1971) d i s c o v e r e d tha t p a r v a l b u m i n s h a v e a h i g h a f f i n i t y f o r c a l c i u m . O r i g i n a l l y i t was t h o u g h t that p a r v a l b u m i n s w e r e c o n f i n e d to w h i t e m u s c l e o f f i s h , a m p h i b i a a n d c e r t a i n rep t i l es ( F o c a n t a n d P e c h e r e , 1965) . A l t h o u g h i t is b a r e l y de tec tab le i n f i s h r e d m u s c l e , P V cons t i tu tes a p p r o x i m a t e l y 0 .7% o f the w e t w e i g h t o f c a r p w h i t e m u s c l e ( G o s s e l i n - R e y , 1974) . S i g n i f i c a n t a m o u n t s o f P V h a v e , h o w e v e r , b e e n f o u n d i n tu r t l e ( L e h k y et a l . , 1974) , c h i c k e n (S t reh le r et a l . , 1977; H e i z m a n n a n d S t r e h l e r , 1979) , r a b b i t ( L e h k y et a l . , 1974; P e c h e r e , 1974) , ra t ( B e r c h t o l d et a l . , 1982a) , ca t ( S t i c h e l et a l . , 1986) , a n d h u m a n ( L e h k y et a l . , 1974; B e r c h t o l d et a l . , 1985) . P a r v a l b u m i n s h a v e no t b e e n i d e n t i f i e d e i t h e r i n i n v e r t e b r a t e t issues o r i n 44 s m o o t h m u s c l e , a n d i t is p resen t o n l y i n l o w a m o u n t s i n c a r d i a c m u s c l e ( G o s s e l i n - R e y , 1974 , B a r o n et a l . , 1975 ; L e P e u c h et a l . , 1979; G e r d a y et a l . , 1979) . E v o l u t i o n T h e e v o l u t i o n a r y h i s t o r y o f p a r v a l b u m i n s d i v i d e s these p r o t e i n s i n t o t w o m a i n g r o u p s , d e s i g n a t e d a a n d f$; b o t h are d e r i v e d f r o m a n a n c e s t r a l gene d u p l i c a t i o n ( G o o d m a n et a l . , 1979) . T h e s e t w o g r o u p s separa te P V a c c o r d i n g to i s o e l e c t r i c p o i n t s , o j - p a r v a l b u m i n ( p H g rea te r t h a n 5.0) b e i n g less a c i d i c t h a n p - p a r v a l b u m i n s . T h e C a 2 + - b i n d i n g p r o t e i n s are t h o u g h t to h a v e e v o l v e d f r o m a f o u r - d o m a i n p r e c u r s o r , b i n d i n g 4 c a l c i u m ions ( G o o d m a n a n d P e c h e r e , 1977) . P a r v a l b u m i n arose b y a d e l e t i o n o f d o m a i n I a n d the d e p r i v a t i o n o f the C a 2 + - b i n d i n g p r o p e r t i e s o f d o m a i n II. C a l m o d u l i n , t r o p o n i n - C a n d the a l k a l i m y o s i n l i gh t c h a i n s are mos t s i m i l a r to the a n c e s t r a l p r o t e i n ( r e v i e w b y G e r d a y , 1982) . B i o c h e m i s t r y o f P a r v a l b u m i n D e s p i t e the p o l y m o r p h i s m o f p a r v a l b u m i n s ( P e c h e r e et a l . , 1971 ; G o s s e l i n - R e y et a l . , 1978) , i t is r e m a r k a b l e h o w cons i s ten t these p r o t e i n s are i n t h e i r p h y s i c a l c h e m i c a l p r o p e r t i e s . P a r v a l b u m i n is a h e a t - s t a b l e a n d w a t e r - s o l u b l e a c i d i c p r o t e i n w i t h an i s o e l e c t r i c p o i n t ( p i ) r a n g i n g f r o m 3.9 i n the c a r p to 6.6 i n the l u n g f i s h , a n d a m o l e c u l a r w e i g h t ( M r ) o f 9 - 13 K D ( G e r d a y et a l . , 1979) . T h e s i ze o f th is p r o t e i n is a lso r e f l e c t e d i n i ts h i g h d i f f u s i o n cons tan t a n d l o w s e d i m e n t a t i o n rate ( P e c h e r e et a l . , 1973) . T h e a m i n o a c i d c o m p o s i t i o n o f P V ( T a b l e II) is c h a r a c t e r i z e d e i t he r b y the absence o r p r e s e n c e o f o n l y one t y r o s i n e o r t r y p t o p h a n r e s i d u e p e r m o l e c u l e , a n d a h i g h n u m b e r o f p h e n y l a l a n i n e res idues a n d a c i d i c a m i n o a c i d s s u c h as aspa r t i c a n d g l u t a m i c a c i d ( P e c h e r e et a l . , 1973) . D u e to the h i g h ra t i o o f p h e n y l a l a n i n e to t y r o s i n e a n d t r y p t o p h a n , P V s h o w s a c h a r a c t e r i s t i c u l t r a v i o l e t a b s o r p t i o n s p e c t r u m i n w h i c h the a b s o r b t i o n b a n d s o f p h e n y l a l a n i n e are v i s i b l e . 45 T A B L E II. A m i n o a c i d c o m p o s i t i o n o f p a r v a l b u m i n i n the rat a n d m o u s e . A m i n o A c i d R a t B r a i n R a t M u s c l e M o u s e M u s c l e L y s i n e 15.5 16.0 16.0 H i s t i d i n e 2.2 2.0 * 1.9 A r g i n i n e 1.0 1.0 1.0 A s p a r t i c a c i d 14.6 14.0 13.8 T h r e o n i n e 5.2 5.0 4 .2 S e r i n e 10.6 11.0 8.1 G l u t a m i c a c i d 9.5 8.0 12.4 P r o l i n e 0.0 0.0 0.9 G l y c i n e 9.4 9.0 9.2 A l a n i n e 11.4 11.0 11.4 V a l i n e 5.2 5.0 6.0 M e t h i o n i n e 2.4 3.0 2.1 I s o l e u c i n e 6.0 6.0 5.9 L e u c i n e 9.6 9.0 9.3 T y r o s i n e 0.0 0.0 0.0 P h e n y l a l a n i n e 8.5 8.0 9.1 T r y p t o p h a n 0.0 0.0 0.0 C y s t e i n e 0.0 0.0 0.0 T h i s tab le has b e e n e x t r a c t e d f r o m d a t a p r e s e n t e d b y H e i z m a n n , 1984. * I nd i ca tes the a m i n o a c i d h i s t i d i n e w h i c h was i o d i n a t e d f o r use i n the R I A d e s c r i b e d o n page 70 . 46 C a l c i u m a n d M a g n e s i u m B i n d i n g T h e bas i c p o l y p e p t i d e s t r u c t u r a l u n i t c o m m o n to P V a n d the o t h e r C a 2 + - b i n d i n g p r o t e i n s s u c h as t r o p o n i n - C a n d c a l m o d u l i n , is an a - h e l i x - C a 2 + - b i n d i n g l o o p - a - h e l i x a n d a n o n h e l i c a l N - t e r m i n u s . O c t a h e d r a l c a l c i u m - b i n d i n g l oops o c c u r b e t w e e n he l i ces C a n d D a n d b e t w e e n h e l i c e s E a n d F . T h e s e s i tes a lso h a v e a h i g h a f f i n i t y f o r m a g n e s i u m . T h e A B d o m a i n is no t C a 2 + - b i n d i n g ( V a n E l d i k et a l . , 1982; W n u k et a l . , 1982) . T h e s t r u c t u r e f o r m e d b y h e l i c e s E a n d F a n d he l i ces C a n d D are c a l l e d the E F a n d C D h a n d s , r e s p e c t i v e l y . T h e p r i m a r y sequence o f P V d i f f e r s f r o m that o f T n - C a n d c a l m o d u l i n . T h e la t te r h a v e f o u r d o m a i n s w h i c h c o n t a i n the bas i c s t r u c t u r a l a - h e l i c a l u n i t . A l l f o u r r eg ions b i n d C a 2 + , a n d i n the case o f T n - C , s i tes 3 a n d 4 have h i g h a f f i n i t y f o r C a 2 + b u t a lso b i n d M g 2 + . A l l o f the c a l c i u m s i tes o n c a l m o d u l i n r e s e m b l e the E F h a n d s o f p a r v a l b u m i n . T h e c a l c i u m - b i n d i n g p r o p e r t i e s o f P V h a v e b e e n e x t e n s i v e l y i n v e s t i g a t e d ( W n u k et a l . , 1982) , a n d these s tud ies h a v e c l a r i f i e d o u r u n d e r s t a n d i n g o f i ts p h y s i o l o g i c a l ro le i n m u s c l e . U n d e r p h y s i o l o g i c a l c o n d i t i o n s (1 m M M g 2 + ) i n the r e s t i n g m u s c l e , m y o p l a s m i c C a 2 + c o n c e n t r a t i o n s are e s t i m a t e d to be 1 0 " 8 M a n d the m a j o r i t y ( 8 0 - 8 5 % ) o f the b i n d i n g s i tes o f P V are o c c u p i e d b y M g 2 + ( C o x et a l . , 1979; P o t t e r et a l . , 1977; R o b e r t s o n et a l . , 1981) . M a g n e s i u m b i n d i n g i n d u c e s a c o n f o r m a t i o n a l c h a n g e at the h y d r o p h o b i c c o r e a n d A B l o o p ( H a i e c h et a l . , 1979a) . A dec rease i n the a f f i n i t y o f P V f o r c a l c i u m i n the p resence o f e l e v a t e d m a g n e s i u m c o n c e n t r a t i o n s is i n d i c a t i v e o f s i m p l e c o m p e t i t i o n f o r the same b i n d i n g s i tes . T h e r e f o r e , i n v i v o . P V is a l w a y s i n a m e t a l - b o u n d state. A s s o c i a t i o n cons tan ts f o r the b i n d i n g o f C a 2 + a n d M g 2 + to P V d i f f e r s l i g h t l y a c c o r d i n g to the m e t h o d o f d e t e r m i n a t i o n , the t y p e o f P V i n v e s t i g a t e d , a n d the c o n d i t i o n s o f m e a s u r e m e n t ( B e n z o n a n a et a l . , 1972; C o x et a l . , 1979; H a i e c h et a l . , 1979b) . T h e a p p a r e n t cons tan ts f o r c a l c i u m range f r o m 1 0 6 to 1 0 9 M " 1 , w h e r e a s the r ange o f v a l u e s f o r m a g n e s i u m is 1 0 4 to 1 0 5 M " 1 . T h e t w o b i n d i n g s i tes are c o n s i d e r e d i n d e p e n d e n t a n d e q u i v a l e n t w i t h respec t to t h e i r m e t a l b i n d i n g p r o p e r t i e s 47 ( C o x et a l . , 1979; C a v e et a l . , 1979; H a i e c h et a l . , 1979a) . E v i d e n c e suggests , h o w e v e r , that the p r o p e r t i e s o f the t w o c a l c i u m si tes a re d i f f e r e n t ( S m i t h a n d W o l e d g e , 1985) . F u n c t i o n a l A s p e c t s A l t h o u g h p a r v a l b u m i n s h a v e b e e n s t u d i e d e x t e n s i v e l y , s i g n i f i c a n c e o f t he i r b i o l o g i c a l f u n c t i o n ( s ) is l i m i t e d . D u e to the h o m o l o g y o f P V w i t h c a l m o d u l i n , t r o p o n i n - C ( T n - C ) a n d m y o s i n l i g h t c h a i n s i t is r easonab le to suggest that the f u n c t i o n o f p a r v a l b u m i n m a y be re l a ted to C a 2 + - a c t i v a t e d p rocesses . H o w e v e r , desp i te the c o m m o n e v o l u t i o n a r y o r i g i n , P V c a n n o t subs t i t u te f o r the o t h e r C a 2 + - b i n d i n g p r o t e i n s ( W n u k et a l . , 1982) . P a r v a l b u m i n has no k n o w n e n z y m a t i c f u n c t i o n s ( C o h e n , 1980; W a l s h et a l . , 1980) , i t does no t i n t e r a c t w i t h t r o p o n i n - I o r t r o p o m y o s i n , n o r c a n i t be p h o s p h o r y l a t e d ( B l u m et a l . , 1977) . I n l i g h t o f the o b s e r v e d m u s c l e t ype s p e c i f i c i t y o f P V , i t has b e e n sugges ted that th is p r o t e i n m a y h a v e a m o r e s p e c i a l i z e d f u n c t i o n i n m u s c l e c o n t r a c t i o n a n d g l y c o l y s i s ( F o c a n t a n d P e c h e r e , 1965; H e i z m a n n , 1984) . H o w e v e r , m a n y o f these f u n c t i o n s h a v e n o w b e e n a t t r i b u t e d to c a l m o d u l i n ( C o h e n , 1980) . C a l m o d u l i n is a s o l u b l e p r o t e i n f o u n d i n m a n y e u k a r y o t e ce l l s . It is p resen t i n b o t h fas t a n d s l o w m u s c l e ( E i b s c h u t z et a l . , 1984) w h e r e i t med ia tes l o n g - t e r m m e t a b o l i c changes s u c h as the b r e a k d o w n o f g l y c o g e n to g l u c o s e . H o w e v e r , i ts ro le i n s k e l e t a l m u s c l e c a l c i u m - t r anspo r t is less w e l l d e f i n e d . T h e ro l e o f p a r v a l b u m i n s r e m a i n e d o b s c u r e u n t i l P e c h e r e a n d c o w o r k e r s (1977) sugges ted that t h e y c o u l d be i n v o l v e d i n the c o n t r a c t i o n - r e l a x a t i o n p rocess o f ske le ta l m u s c l e . B r i g g s (1975) h a d e a r l i e r sugges ted that P V is the ' s o l u b l e r e l a x i n g f a c t o r ' o f m u s c l e . C a l c i u m f r e e P V c a n take u p c a l c i u m f r o m T n - C ( P e c h e r e et a l . , 1977) a n d t h e r e b y c o m p l e t e l y i n a c t i v a t e the A T P a s e a c t i v i t y o f the m y o f i b r i l s ( G e r d a y a n d G i l l i s , 1976) , w h i l e f r a g m e n t e d S R w i l l r e t u r n P V to the c a l c i u m - f r e e state ( G e r d a y a n d 48 G i l l i s , 1976; B l u m et a l . , 1977) . P a r v a l b u m i n , t h e r e f o r e , p r o b a b l y w o r k s as the shu t t l e m e c h a n i s m f o r C a 2 + b e t w e e n the m y o f i b r i l a n d the S R d u r i n g m u s c l e r e l a x a t i o n ( P e c h e r e , et a l . , 1977) . A s c h e m e i l l u s t r a t i n g the m o v e m e n t o f C a 2 + i n fas t ske le ta l m u s c l e ( F i g . 17) was d e v e l o p e d b y P e c h e r e et a l (1977) a n d la te r m o d i f i e d b y H e i z m a n n (1984) . W h e n the m u s c l e c e l l is a c t i v a t e d to re lease c a l c i u m f r o m the S R , the l i b e r a t e d i o n is no t • t r a p p e d b y P V i m m e d i a t e l y b u t i n te rac ts w i t h t r o p o n i n - C . T h i s s e q u e n c e o f even ts is poss ib l e d u e to the d i f f e r e n t b i n d i n g k i n e t i c s o f P V a n d T n - C ( H a i e c h et a l . , 1979a) . A l t h o u g h the a f f i n i t y o f p a r v a l b u m i n f o r C a 2 + is s i m i l a r to that o f T n - C , the rate o f b i n d i n g o f T n - C exceeds that o f P V . A s w e l l , i n the c a l c i u m - f r e e state the b i n d i n g s i tes o n the P V m o l e c u l e are o c c u p i e d b y m a g n e s i u m ( M g 2 + ) w h i c h m u s t f i r s t d i s soc i a t e to a l l o w C a 2 + - b i n d i n g . T h e d i s s o c i a t i o n o f M g 2 + a n d s u b s e q u e n t u p t a k e o f C a 2 + b y p a r v a l b u m i n is the ra te l i m i t i n g s tep , m a k i n g p o s s i b l e the b i n d i n g o f C a 2 + to T n - C w h i c h t r i ggers c o n t r a c t i o n ( H a i e c h et a l . , 1979a). C a l c i u m b i n d i n g to P V is d e p e n d e n t o n the M g 2 + c o n c e n t r a t i o n a n d o n t e m p e r a t u r e ( O g a w a a n d T a n o k u r a , 1986) . T h e a c t u a l d i s t r i b u t i o n o f c a l c i u m b e t w e e n the P V a n d T n - C b i n d i n g s i tes , as s t u d i e d b y c o m p u t e r s i m u l a t i o n ( G i l l i s , 1980; G i l l i s et a l . , 1982) , is s u c h that 9 7 % o f the c a t i o n b i n d i n g s i tes o f T n - C are sa tu ra ted at the t i m e o f a C a 2 + p u l s e ( R o b e r t s o n et a l . , 1981) . C o n v e r s e l y , the t r a n s f e r o f C a 2 + to the b i n d i n g s i tes o f P V is a c h i e v e d i n a p p r o x i m a t e l y 2 0 0 m s . I n i t i a l l y f o l l o w i n g m u s c l e r e l a x a t i o n , m u c h o f the c a l c i u m is s t i l l p a r v a l b u m i n - b o u n d a n d w i l l be t r a n s p o r t e d b a c k to the S R d u r i n g the r e c o v e r y p e r i o d ( G i l l i s , 1980) . H e n c e , i t has b e e n p r o p o s e d that the b i o l o g i c a l s i g n i f i c a n c e o f P V is to a l l o w fas t r e l a x a t i o n o f m u s c l e because o f i ts r e l a x i n g e f f e c t d u e to the b i n d i n g o f c a l c i u m ( A s h l e y a n d G r i f f i t h s , 1983; G i l l i s 1980; G i l l i s et a l . , 1985) . T h e p r e c e d i n g h y p o t h e s i s has b e e n q u e s t i o n e d o n a n u m b e r o f a c c o u n t s . F i r s t l y , the k i n e t i c s suggest that the s l o w o f f rate o f b o t h C a 2 + a n d M g 2 + p r e v e n t s P V f r o m b e i n g d i r e c t l y i n v o l v e d i n the r a p i d d e c a y o f t e n s i o n a f t e r a s i ng le t w i t c h ( J o h n s o n et a l . , 1981 ; R o b e r t s o n et a l . , 1981) . S e c o n d l y , i n the e v e n t o f a t e tanus , P V w o u l d 49 F i g u r e 17 S c h e m a t i c d i a g r a m i l l u s t r a t i n g the p r o p o s e d f u n c t i o n o f p a r v a l b u m i n ( P V ) i n f a s t - t w i t c h m u s c l e . I n the r e l a x e d state the c a l c i u m - b i n d i n g s i tes are b o u n d b y m a g n e s i u m ( M g 2 ) a n d c a l c i u m ( C a 2 + ) is s t o red i n the s a r c o p l a s m i c r e t i c u l u m ( S R ) . U p o n a r r i v a l o f a n a c t i o n p o t e n t i a l , C a 2 + is re leased f r o m the S R a n d t a k e n u p b y the f i r s t a v a i l a b l e b i n d i n g s i tes w h i c h are o n the t r o p o n i n - C m o l e c u l e o f the m y o f i b r i l s ( m f ) . B e c a u s e o f the h i g h a f f i n i t y o f P V f o r C a 2 + , m a g n e s i u m is d i s p l a c e d f r o m P V a n d C a 2 + t h e n b i n d s to the f r e e s i tes . P V is t h e n t h o u g h t to t r anspo r t C a 2 + b a c k to the S R at w h i c h p o i n t M g a g a i n b i n d s to the f r e e s i tes a n d the m u s c l e re laxes ( A d o p t e d f r o m H e i z m a n n , 1984) . 50 RELAXED STATE CONTRACTION action potential PVCa 2 TRANSITIONAL STATE 57 q u i c k l y b e c o m e sa tu ra ted w i t h c a l c i u m because o f the s l o w d i s s o c i a t i o n rate a n d t h e r e b y d i m i n i s h i ts r e l a x i n g e f f e c t ( G e r d a y , 1982; O g a w a a n d T a n o k u r a , 1986) . T h e s e q u e r i e s c o n t i n u e to d e m a n d a m o r e p r e c i s e e x p l a n a t i o n f o r the ro le o f P V i n m u s c l e c o n t r a c t i o n / r e l a x a t i o n . T h e m e t a l - b i n d i n g p r o p e r t i e s o f c a l m o d u l i n a re s i m i l a r to those o f the c a l c i u m - s p e c i f i c s i tes o f t r o p o n i n - C (Po t te r et a l . , 1977) . C a l m o d u l i n c o n t a i n s 4 C a 2 + - s p e c i f i c s i tes , n o n e o f w h i c h b i n d M g 2 + . T h e a s s o c i a t i o n cons tan ts a n d rate o f b i n d i n g f o r the c a l c i u m - s i t e s o f c a l m o d u l i n are s i m i l a r to those o f t r o p o n i n - C , h o w e v e r , the c o n c e n t r a t i o n o f c a l m o d u l i n i n ske le ta l m u s c l e is m u c h l o w e r t h a n tha t o f t r o p o n i n ( R o b e r t s o n et a l . , 1981) . M a t h e m a t i c a l m o d e l s e x p r e s s i n g the t i m e - c o u r s e o f C a 2 + - b i n d i n g to c a l m o d u l i n , t r o p o n i n , p a r v a l b u m i n a n d m y o s i n i n r esponse to t rans ien t i nc reases i n f r e e m y o p l a s m i c c a l c i u m c o n c e n t r a t i o n s ( p C a ) , i n d i c a t e that a l l C a 2 + - s i t e s b i n d c a l c i u m i n response to a s i ng le p C a t rans ien t ( R o b e r t s o n et a l . , 1981) . H o w e v e r , o n l y the C a 2 + - s p e c i f i c s i tes o f t r o p o n i n a n d c a l m o d u l i n b e c o m e a d e q u a t e l y sa tu ra ted fas t e n o u g h to p l a y a ro le i n r a p i d c a l c i u m r e g u l a t i o n o f c o n t r a c t i o n . T h e c a l c i u m c o n t e n t o f the C a 2 + - M g 2 + - s i t e s o f t r o p o n i n , p a r v a l b u m i n , a n d m y o s i n i nc reases o n l y s l i g h t l y . M o r e o v e r , e x c h a n g e o f c a l c i u m o n t o a n d o f f e a c h c lass o f s i tes , ana logous to a c o n t r a c t i o n - r e l a x a t i o n c y c l e , o n l y o c c u r r e d w i t h the C a 2 + - s p e c i f i c s i tes o f t r o p o n i n a n d c a l m o d u l i n . P r e s e n t i n the 2 - 0 . 0 5 m M r a n g e , P V ex is ts i n adequa te q u a n t i t i e s to b i n d a l l the c a l c i u m o f the s a r c o p l a s m ( A s h l e y , 1983) . A t 2 0 ° C , a n e s t i m a t e d 330 u m o l o f C a 2 + / l i t e r is b o u n d to P V at rest ( O g a w a a n d T a n o k u r a , 1986) . T h i s i nc reases to 6 5 0 - 6 7 0 p m o l C a 2 + / l i t e r d u r i n g a te tanus a n d is f u r t h e r a c c e n t u a t e d w h e n the t e m p e r a t u r e is l o w e r e d ( O g a w a a n d T a n o k u r a , 1986) . S i n c e the b i n d i n g s i tes o n P V a p p e a r to h a v e a h i g h e r a f f i n i t y f o r c a l c i u m b u t a s l o w e r a s s o c i a t i o n rate t h a n f o r the r e g u l a t o r y s i tes o n t r o p o n i n ( H a i e c h et a l . , 1979a) , the a m o u n t o f c a l c i u m r e q u i r e d to ac t i va te f a s t - t w i t c h m u s c l e w h e n P V is p resen t is l o w e r t h a n w o u l d be p r e d i c t e d b y the m y o f i b r i l l a r C a 2 + - b i n d i n g c a p a c i t y ( R o b e r t s o n et a l . , 1981) . T h e c a l c u l a t e d g a i n i n 52 c a l c i u m b y P V a n d d i s s o c i a t i o n b y T n - C f o l l o w i n g a s t i m u l a t i o n , i n d i c a t e s that 2 ou t o f 3 C a 2 + i ons that l eave t r o p o n i n are t e m p o r a r i l y b o u n d to P V b e f o r e b e i n g seques te red b y the S R ( B a y l o r et a l . , 1983) . I n a d d i t i o n , the m a x i m u m ra te at w h i c h the S R m u s t f u n c t i o n to r e m o v e c a l c i u m f r o m the m y o f i b r i l s is d e c r e a s e d b y m o r e t h a n 5 0 % ( B a y l o r et a l . , 1983) , f u r t h e r s u p p o r t i n g the ro le o f P V as a so lub le r e l a x i n g - f a c t o r i n f a s t - t w i t c h m u s c l e . A d i s t u r b a n c e i n the f u n c t i o n a l c a p a c i t y o f p a r v a l b u m i n m a y , t h e r e f o r e , h a v e se r ious i m p l i c a t i o n s f o r the r e g u l a t i o n o f i n t r a c e l l u l a r c a l c i u m a n d m u s c l e c o n t r a c t i o n . I n t r a c e l l u l a r D i s t r i b u t i o n P a r v a l b u m i n s are no t r e s t r i c t e d to m u s c l e t issue a l o n e . R e c e n t l y , t h e i r p resence has b e e n r e p o r t e d i n n e r v o u s t issue ( G o s s e l i n - R e y , 1974: C e l i o a n d H e i z m a n n , 1981; B e r c h t o l d et a l 1985; G e r f e n et a l . , 1985; E n d o et a l . , 1986b ) , a n d i n o t h e r t issues s u c h as the s k i n , tes t is , s p l e e n , k i d n e y , b o n e , t ee th , a n d the o v a r y ( B e r c h t o l d et a l . , 1984) . S u c h a d i v e r s e d i s t r i b u t i o n i n d i c a t e s that P V m a y h a v e m u l t i p l e f u n c t i o n s assoc ia ted w i t h v a r i o u s c a l c i u m - d e p e n d e n t p rocesses . T h e d i s t r i b u t i o n o f P V , p r i m a r i l y d e t e r m i n e d u s i n g i m m u n o l o g i c a l m e t h o d s , is s u m m a r i z e d i n T a b l e III o n page 57 . S tud ies h a v e s h o w n that P V has a r es t r i c t ed i m m u n o l o g i c a l response i n that c r o s s - r e a c t i v i t y is l i m i t e d to c l o s e l y r e l a t ed spec ies . In a d d i t i o n , no c r o s s - r e a c t i v i t y has b e e n o b s e r v e d b e t w e e n d i f f e r e n t i so t ypes o f P V , o r w i t h the o t h e r C a 2 + - b i n d i n g p r o t e i n s ( G e r d a y , 1982) . It is t h e r e f o r e i m p o r t a n t w h e n m a p p i n g the d i s t r i b u t i o n o f th is p r o t e i n b y i m m u n o l o g i c a l m e a n s , to use an t i se ra s p e c i f i c to a p a r t i c u l a r spec ies . E a r l y s tud ies o f P V c o n c l u d e d that i ts l o c a l i z a t i o n was r e s t r i c t e d to w h i t e m u s c l e o f f i s h a n d a m p h i b i a n s ( F o c a n t a n d P e c h e r e , 1965) . A d e c a d e l a te r , e v i d e n c e was p r e s e n t e d i n d i c a t i n g that the d i s t r i b u t i o n o f P V i n c l u d e d t issues o f h i g h e r ve r teb ra tes as w e l l ( L e h k y et a l . , 1974; P e c h e r e , 1974) . M o r e r e c e n t l y i t has b e c o m e c l e a r that p a r v a l b u m i n s are p resen t to s o m e degree i n a l l ve r teb ra tes i n c l u d i n g m a n ( r e v i e w b y 53 W n u k et a l . , 1982) . E v e n t h o u g h p a r v a l b u m i n s are essen t i a l l y m u s c l e p r o t e i n s , t h e i r q u a n t i t y a n d d i s t r i b u t i o n d i f f e r a c c o r d i n g to m u s c l e t y p e . H i g h P V con ten ts a re f o u n d i n w h i t e m u s c l e , ( G o s s e l i n - R e y a n d G e r d a y , 1977) w h i l e l o w l eve l s a re n o t e d i n r e d musc les w i t h h i g h m y o g l o b i n con ten ts ( H a m o i r et a l . , 1980) . W i t h r e g a r d to f i b e r t y p e , P V is a b u n d a n t i n t y p e 2 f i b e r s bu t v i r t u a l l y absent i n t y p e 1 e x t r a f u s a l f i b e r s w h e n c o m p a r e d to s t a n d a r d m y o f i b r i l l a r A T P a s e s t a i n i n g ( C e l i o a n d H e i z m a n n , 1982) . W i t h i n the t y p e 2 c a t e g o r i z a t i o n o f f i b e r s ( types 2 A , 2 B , a n d 2 C ) there is a c o n t i n u u m o f P V c o n c e n t r a t i o n s ( C e l i o a n d H e i z m a n n , 1982) , c l o s e l y f o l l o w i n g the a e r o b i c a n d a n a e r o b i c m e t a b o l i s m i n these ce l l s . T h i s h i s t o c h e m i c a l f i n d i n g is s u p p o r t e d b y e l e c t r o p h o r e t i c a n a l y s i s o f t y p e d s i ng l e m u s c l e f i b e r s ( H e i z m a n n et a l . , 1982) . O n l y those f i b e r s j u d g e d as t y p e 2 B / F G p r o d u c e d a la rge P V spo t at the c h a r a c t e r i s t i c m o l e c u l a r w e i g h t a n d i s o e l e c t r i c p o i n t o n t w o - d i m e n s i o n a l ge ls . N o spot c o u l d be i d e n t i f i e d o n the gels f r o m t y p e 1 / S O f i b e r s . T y p e 2 A f i b e r s w i t h f a s t - o x i d a t i v e - g l y c o l y t i c p r o p e r t i e s v a r y i n t h e i r P V c o n t e n t . H o w e v e r , there are a f e w e x c e p t i o n s to th is c a t e g o r i z a t i o n . S i g n i f i c a n t a m o u n t s o f P V are p resen t i n r e d m u s c l e s ( w i t h p r e d o m i n a n t l y s l o w - o x i d a t i v e f i b e r s ) o f the e l e c t r i c ee l ( C h i l d e r s a n d S i e g e l , 1976) a n d c a r p ( G o s s e l i n - R e y et a l . , 1978) . I n the c h i c k e n , o n the o t h e r h a n d , P V is absen t f r o m w h i t e breas t m u s c l e ( L e P e u c h et a l . , 1979) . T h e r e f o r e , the r e l a t i o n s h i p b e t w e e n h i g h P V a n d l o w m y o g l o b i n c o n t e n t ( G e r d a y et a l . , 1979) does no t h o l d f o r a l l spec ies . M o r e c o n v i n c i n g is the c o r r e l a t i o n o f P V i m m u n o r e a c t i v i t y w i t h r e l a x a t i o n t i m e a n d the d e v e l o p m e n t o f the S R ( C e l i o a n d H e i z m a n n , 1982) . P a r v a l b u m i n a n t i s e r a reac t the s t ronges t t o w a r d f i b e r s w i t h the fastest r e l a x a t i o n t imes a n d c o n s e q u e n t l y the mos t w e l l d e v e l o p e d S R , s u c h as that i n t ype 2 / F G f i b e r s . A c o n t i n u u m o f P V s t a i n i n g i n tens i t i es resu l ts as these t w o p r o p e r t i e s c h a n g e w i t h i n the m u s c l e . I n m u s c l e s p i n d l e s , P V has b e e n l o c a l i z e d i n one o f the n u c l e a r c h a i n i n t r a f u s a l f i b e r s b u t i n n o n e o f the n u c l e a r b a g f i b e r s ( C e l i o a n d H e i z m a n n , 1982) . T h e s e w o r k e r s sugges ted that the i n t r a f u s a l c h a i n f i b e r p r o b a b l y has fas te r r e l a x i n g 54 p r o p e r t i e s t h a n d o the bag f i b e r s o r the n o n - r e a c t i v e c h a i n f i b e r . A c o m p r e h e n s i v e s t u d y o f the r e g i o n a l d i s t r i b u t i o n o f P V i n i n t r a f u s a l f i b e r s does no t ex i s t at th is t i m e . O f the n o n - m u s c l e t issues e x a m i n e d , the c e r e b e l l u m a n d n e o c o r t e x o f the b r a i n c o n t a i n s i g n i f i c a n t quan t i t i e s o f P V ( T a b l e III). T h e P V f o u n d i n m a n y o f these n o n - m u s c l e t issues c r o s s - r e a c t s w i t h m u s c l e a n t i - P V s e r u m ( H e i z m a n n , 1984) . R a t b r a i n P V , the o n l y n o n - m u s c l e P V that has b e e n f u l l y i so l a t ed a n d c h a r a c t e r i z e d ( B e r c h t o l d et a l . , 1982b , is i n d i s t i n g u i s h a b l e b i o c h e m i c a l l y a n d i m m u n o l o g i c a l l y f r o m m u s c l e P V . P a r v a l b u m i n appea rs to be a s o l u b l e p r o t e i n , f r e e l y d i s p e r s e d t h r o u g h o u t the c y t o s o l . I n d i r e c t i m m u n o f l u o r e s c e n c e a p p l i e d to c a r p m u s c l e ( B e n z o n a n a et a l . , 1975 , 1977) has s h o w n a u n i f o r m d i s t r i b u t i o n o f P V i n m y o f i b r i l s w i t h no s p e c i f i c l o c a l i z a t i o n to a n y p a r t i c u l a r i n t r a c e l l u l a r o r g a n e l l e . I n s k i n n e d m u s c l e f i b e r s , P V w i l l d i f f u s e c o m p l e t e l y ou t o f the ce l l s ( G i l l i s et a l . , 1979) . T h e d a t a f r o m these m o r p h o l o g i c a l a n d b i o c h e m i c a l s tud ies i n d i c a t e that P V is no t assoc ia ted w i t h a n y p a r t i c u l a r s t ruc tu re (s ) w i t h i n the c e l l e v e n t h o u g h i ts r e l a t i o n s h i p w i t h C a 2 + - r e g u l a t i o n suggests that i t m i g h t be . U l t r a s t r u c t u r a l l o c a l i z a t i o n o f P V i n ce l l s o f the b r a i n is i n c o n c l u s i v e ( H e i z m a n n a n d C e l i o , 1986) . P r e - e m b e d d i n g s t a i n i n g t e c h n i q u e s u s e d to s t u d y P V i n z e b r a f i n c h ( Z u s c h r a t t e r et a l . , 1985) a n d rat ( H e i z m a n n a n d C e l i o , 1986) , r e v e a l e d l a b e l l i n g assoc ia ted w i t h m i c r o t u b u l e s , i n t r a c e l l u l a r m e m b r a n e s a n d p o s t s y n a p t i c dens i t i es . P o s t - e m b e d d i n g t e c h n i q u e s p e r f o r m e d o n rat c e r e b e l l u m ( H e i z m a n n a n d C e l i o , 1986) c o n c l u d e d that P V was i n d e e d h o m o g e n e o u s l y d i s p e r s e d t h r o u g h o u t the c y t o p l a s m o f the c e l l . S i m i l a r k i n d s o f s tud ies h a v e no t b e e n p u b l i s h e d o n m u s c l e o r o the r n o n - m u s c l e t i ssue . Q u a n t i t a t i o n o f P a r v a l b u m i n P a r v a l b u m i n has b e e n i d e n t i f i e d i n e v e r y m a m m a l i a n spec ies s t u d i e d , a n d i t has b e e n l o c a l i z e d i n a w i d e v a r i e t y o f ce l l s b y i m m u n o h i s t o c h e m i c a l t e c h n i q u e s 55 ( H e i z m a n n , 1984) . Q u a n t i t a t i v e l y , i ts c o n t e n t d i f f e r s c o n s i d e r a b l y b e t w e e n i n d i v i d u a l ce l l s o r b e t w e e n t issue t ypes as w e l l as w i t h i n p a r t i c u l a r t i ssues. E s t i m a t i o n s o f the a m o u n t o f P V c o n t a i n e d w i t h i n these v a r i o u s t issues has a i d e d i n the u n d e r s t a n d i n g o f i ts b i o l o g i c a l s i g n i f i c a n c e . T a b l e III s u m m a r i z e s the v a r i o u s t issues e x a m i n e d to date a n d t h e i r c o r r e s p o n d i n g P V con ten t . R e p o r t s o f P V c o n c e n t r a t i o n s m a y v a r y s o m e w h a t d u e to the m e t h o d o f d e t e c t i o n . Ins tead o f the m o r e c o n v e n t i o n a l m e t h o d s , h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y ( H P L C ) is n o w c o m m o n l y u s e d f o r s m a l l q u a n t i t i e s o f t issue because o f the h i g h p e r c e n t y i e l d o b t a i n e d w i t h th is t e c h n i q u e . I n a d d i t i o n , H P L C is a sens i t i ve a n d e f f i c i e n t t oo l f o r p e p t i d e ana l ys i s ( B e r c h t o l d et a l . , 1983) . I m m u n o c h e m i c a l q u a n t i f i c a t i o n o f P V has a lso b e e n s u c c e s s f u l b y r e c e n t l y d e v e l o p e d R I A p r o c e d u r e s ( B e r c h t o l d et a l . , 1985 ; E n d o et a l . , 1985a , 1985b) a n d E L I S A m e t h o d s ( L e b e r e r a n d P e t t e , 1986a , 1986b) . T h e a i m o f th is s e c t i o n o f the thes is was to d o c u m e n t the changes i n the c o n c e n t r a t i o n a n d d i s t r i b u t i o n o f P V i n i n t r a f u s a l a n d e x t r a f u s a l m u s c l e f i b e r s o f the f a s t - t w i t c h E D L a n d the s l o w - t w i t c h so leus m u s c l e s o f n o r m a l a n d g e n e t i c a l l y d y s t r o p h i c m i c e b y b i o c h e m i c a l a n d i m m u n o c h e m i c a l t e c h n i q u e s . B e c a u s e m u s c u l a r d y s t r o p h y is a p r o g r e s s i v e d isease a n d P V c o n t e n t appea rs to be d e v e l o p m e n t a l l y r e g u l a t e d , th is s t u d y was c o n d u c t e d o n m i c e at 2 a n d 32 w e e k s o f age . A n i m a l s at 2 - w e e k s o f age w e r e c h o s e n because c l i n i c a l s igns o f d y s t r o p h y h a v e no t ye t a p p e a r e d a n d b y 3 2 - w e e k s o f age the d isease has r e a c h e d an a d v a n c e d state. A n a n t i s e r u m to m o u s e P V was r a i s e d i n r abb i t s a n d used f o r i m m u n o l o g i c a l l o c a l i z a t i o n o f P V at the l i gh t m i c r o s c o p i c l e v e l . S i n c e P V has b e e n s h o w n to be l o c a l i z e d i n s p e c i f i c f i b e r t y p e s , a n d s i n c e a l t e ra t i ons i n f i b e r t ypes are a c o m m o n f e a t u r e o f m u s c u l a r d y s t r o p h y , a c o r r e l a t i o n b e t w e e n P V a n d f i b e r t y p e was c o n d u c t e d u s i n g a m o d i f i c a t i o n o f the c o n v e n t i o n a l m y o s i n A T P a s e s t a i n i n g p r o c e d u r e . 56 T A B L E III. S u m m a r y o f p a r v a l b u m i n c o n t e n t i n v a r i o u s spec ies a n d t i ssue. Spec ies / T i s s u e R e f e r e n c e P V C o n t e n t M i c e g a s t r o c n e m i u s so leus E D L H e i z m a n n et a l . , 1982 ( H P L C ) 4 .9 g / k g w e t w t 0.01 4.4 T i b i a l i s A n t e r i o r g a s t r o c n e m i u s T i b i a l i s A n t e r i o r q u a d r i c e p s D y s t r o p h i c M i c e g a s t r o c n e m i u s T i b i a l i s A n t e r i o r q u a d r i c e p s b r a i n c u l t u r e s R a t g a s t r o c n e m i u s so leus E D L T i b i a l i s A n t e r i o r b r a i n ( to ta l ) c e r e b e l l u m c e r e b r u m c e r e b e l l u m c e r e b r u m r e t i n a l u n g , l i v e r , s p l e e n p ros ta te b o n e test is s k i n K l u g et a l . , 1985 (e l ec t ropho res i s ) K l u g et a l . , 1985 P f y f f e r et a l . , 1984 H e i z m a n n et a l . , 1982 S c h n e e b e r g e r et a l . , 1985 ( H P L C & R I A ) B e r c h t o l d et a l . , 1984 ( H P L C ) E n d o et a l 1986b ( R I A ) B e r c h t o l d et a l . , 1984 ( H P L C ) 4.8 5.53 m g / g w e t w t 4 .86 5.82 3.24 m g / g w e t w t 2.98 3.44 5 % to ta l heat s tab le p r o t e i n 3.3 g / k g w e t w t 0 .004 2.4 2.7 0 .02 g / k g w e t w t 15 m g / k g w e t w t 2 3217 n g / m g T S P 128 1710 10 24 m g / k g w e t w t 15 11 86 H o r s e d e e p g lu teus M a n vas tus , t r i c e p s G u i n e a P i g so leus sa r t o r i us H e i z m a n n et a l . , 1982 0.001 g / k g we t 0.001 0 .007 0 .25 R a b b i t E D L so leus vas tus ( s u p e r f i c i a l i s ) L e b e r e r & P e t t e , 1986a ( E L I S A ) 544 p g / g w e t 4.4 1200 57 M A T E R I A L A N D M E T H O D S G e n e r a l N o r m a l (+/+) a n d d y s t r o p h i c ( d y 2 J / d y 2 J ) a d u l t m a l e a n d n e o n a t a l m a l e o r f e m a l e m i c e o f the C 5 7 B L / 6 J s t r a i n w e r e o b t a i n e d f r o m o u r o w n m o u s e c o l o n i e s . O r i g i n a l b r e e d i n g p a i r s w e r e p u r c h a s e d f r o m the J a c k s o n L a b o r a t o r i e s , B a r H a r b o u r , M E a n d m a i n t a i n e d b y the c r o s s - i n t e r c r o s s s y s t e m ( F l a h e r t y , 1981) . S i b l i n g +/+ m i c e w e r e used as c o n t r o l s a n d h o m o z y g o u s d y 2 J / d y 2 J a n i m a l s w e r e u s e d as d y s t r o p h i c s f o r a l l e x p e r i m e n t s . S o m e o f the n o r m a l m i c e w e r e b a c k c r o s s e d w i t h d y 2 J / d y 2 J d y s t r o p h i c m i c e r e s u l t i n g i n + / d y 2 J he te rozygo tes . H e t e r o z y g o t e pa i r s w e r e i n t e r c r o s s e d f o r the p r o d u c t i o n o f d y 2 J / d y 2 J p r o g e n y . T h i s s y s t e m was s u c c e s s f u l f o r a l l a d u l t m i c e used i n the p r o j e c t . S o m e m o d i f i c a t i o n s to th is b r e e d i n g s y s t e m w e r e necessa ry f o r the neona ta l age g r o u p . C l i n i c a l s igns o f m u s c u l a r d y s t r o p h y are no t m a n i f e s t e d i n the y o u n g m i c e o f th is s t r a i n u n t i l 3 - 4 w e e k s ( A t w o o d a n d K w a n , 1978) so that the use o f he te rozygo te i n t e r c ross o f f s p r i n g is u n r e l i a b l e f o r a n i m a l s less t h a n 3 w e e k s o f age . T h e r e f o r e , o n l y h o m o z y g o t e m a t i n g s w e r e used f o r m i c e o f 1 a n d 2 w e e k s o f age . T h i s d y s t r o p h i c m u r i n e s t r a i n is no t a p r o l i f i c b reede r . M a l e s a re o f t e n i n f e r t i l e a n d h a v e d i f f i c u l t y m o u n t i n g t h e i r m a t e s , w h e r e a s f e m a l e s o f t e n have p r o b l e m s d e l i v e r i n g t h e i r o f f s p r i n g ( Y o u n g e r a n d S i l v e r m a n , 1984) . M e t h o d s w e r e u n d e r t a k e n to o b t a i n d y 2 J / d y 2 J d y s t r o p h i c a n i m a l s , a n d i n c l u d e d o v a r i a n t r a n s p l a n t a t i o n ( Jones a n d K r o h n , 1960; T a n i o k a et a l . 1973) a n d a r t i f i c i a l i n s e m i n a t i o n ( W o l f e a n d S o u t h a r d , 1962) t e c h n i q u e s . I n a d d i t i o n , h i n d l i m b a m p u t a t i o n s w e r e p e r f o r m e d to i d e n t i f y m i c e o f the a p p r o p r i a t e g e n o t y p e i n the 2 - w e e k age g r o u p . T w o - w e e k - o l d m i c e w e r e o b t a i n e d f r o m a cross b e t w e e n h e t e r o z y g o t e ma les (+ /dy 2 ^ ) a n d n o r m a l f e m a l e m i c e c a r r y i n g o v a r i e s t r a n s p l a n t e d f r o m h o m o z y g o t e f e m a l e s ( d y 2 J / d y 2 J ) . A n i m a l s w e r e a n e s t h e t i z e d w i t h e the r , a n d the r i g h t l eg f r o m e a c h 2 - w e e k - o l d h e t e r o z y g o t e ( + / d y 2 J ) m o u s e was a m p u t a t e d p r o x i m a l to the k n e e 58 j o i n t . T h e E D L a n d so leus m u s c l e s w e r e s u b s e q u e n t l y d i s s e c t e d ou t a n d p r o c e s s e d a c c o r d i n g to the m e t h o d s d e s c r i b e d f o r i m m u n o h i s t o c h e m i c a l a n d h i s t o c h e m i c a l a n a l y s i s . A t the t i m e o f s u r g e r y , the f r e e e n d i n g s o f the m a j o r b l o o d vessels w e r e t i e d , the s k i n was s u t u r e d a n d c l a m p e d , a n d m i c e w e r e a l l o w e d to r e c o v e r u n d e r a heat l a m p b e f o r e r e t u r n i n g t h e m to t h e i r m o t h e r s . A t 3 w e e k s o f age , the m i c e w e r e c h e c k e d f o r c l i n i c a l s igns o f d y s t r o p h y . A n i m a l s w e r e c o n s i d e r e d d y s t r o p h i c a c c o r d i n g to the f o l l o w i n g tests: s p l a y i n g a n d c l e n c h i n g o f the l e f t h i n d leg a n d f o o t , i n a b i l i t y to i m m e d i a t e l y r e c o v e r b a l a n c e a f t e r b e i n g s p u n b y the t a i l , a n d i n a b i l i t y to g rasp the r u n g s o f the cage top w i t h the l e f t f o o t w h i l e c l i m b i n g the r u n g s o r w h i l e the cage top was s u s p e n d e d u p s i d e d o w n . O u t o f a to ta l o f 34 a m p u t a t i o n s , 31 a n i m a l s s u r v i v e d the s u r g e r y a n d 14 o f these w e r e j u d g e d to be d y s t r o p h i c . F r o m th is g r o u p , 10 m i c e w e r e u s e d f o r the i m m u n o h i s t o c h e m i s t r y e x p e r i m e n t s . A g e n e r a l o u t l i n e f o r the m e t h o d o l o g y i n v o l v e d i n the i m m u n o c y t o c h e m i c a l a n d b i o c h e m i c a l aspec ts o f th is thes is w o r k is d e p i c t e d i n F i g u r e 18. T h e s a m p l e s ize (n) f o r e a c h e x p e r i m e n t a l g r o u p was 5 m i c e , un less o t h e r w i s e s p e c i f i e d . P a r v a l b u m i n was c h a r a c t e r i z e d i n b o t h age g r o u p s o n o n e - d i m e n s i o n a l s o d i u m d o d e c y l su l f a te ( S D S ) s lab gels f r o m w h i c h the p r o t e i n s w e r e e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d to n i t r o c e l l u l o s e p a p e r a n d i m m u n o b l o t t e d w i t h the P V an t i se ra . P a r v a l b u m i n c o n t e n t was assessed b y a r a d i o i m m u n o a s s a y ( R I A ) d e v e l o p e d i n D r . K . B a i m b r i d g e s ' s l a b o r a t o r y i n the D e p a r t m e n t o f P h y s i o l o g y , T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a . T h e i m m u n o h i s t o c h e m i c a l l o c a l i z a t i o n o f P V was a c c o m p l i s h e d b y the s t a n d a r d p e r o x i d a s e - a n t i p e r o x i d a s e m e t h o d o l o g y . H i s t o c h e m i c a l f i b e r t y p i n g f o r m y o f i b r i l l a r A T P a s e was c o n d u c t e d a c c o r d i n g to a m o d i f i c a t i o n o f the B r o o k e a n d K a i s e r (1970) m e t h o d u t i l i z i n g m e t a c h r o m a t i c t o l u i d i n e b l u e ( D o r i g u z z i et a l . , 1983) . C h a r a c t e r i z a t i o n o f P a r v a l b u m i n P a r v a l b u m i n was i n i t i a l l y c h a r a c t e r i z e d i n samp les o f E D L a n d so leus m u s c l e s o f 3 2 - w e e k a n d 2 - w e e k - o l d m i c e b y the t w o - d i m e n s i o n a l ge l e l e c t r o p h o r e s i s m e t h o d o f 59 F i g u r e 18 S c h e m a t i c i l l u s t r a t i o n o u t l i n i n g the g e n e r a l m e t h o d o l o g y f o l l o w e d i n th is p o r t i o n o f the s t u d y . A . P a r v a l b u m i n f r o m the f a s t - t w i t c h m u s c l e o f n o r m a l m i c e was i so la ted o n i s o e l e c t r i c f o c u s i n g ( I E F ) t ube ge ls . T h e b a n d was e x c i s e d a n d e m u l s i f i e d i n P B S a n d F r e u n d ' s c o m p l e t e a d j u v a n t , a n d th is s o l u t i o n was i n j e c t e d i n t o r a b b i t s . T h e a n t i s e r a (1 A B ) c o l l e c t e d was tested f o r p a r v a l b u m i n s p e c i f i c i t y a n d t h e n u s e d f o r i m m u n o c y t o c h e m i s t r y ( I C C ) , r a d i o i m m u n o a s s a y s ( R I A ) a n d i m m u n o b l o t s . B . T h e a c t u a l e x p e r i m e n t s i n v o l v e d r e m o v i n g the ex tenso r d i g i t o r u m l o n g u s ( E D L ) a n d so leus ( S O L ) f r o m b o t h n o r m a l a n d d y s t r o p h i c a d u l t a n d neona ta l m i c e . T h e m u s c l e s w e r e e i t he r h o m o g e n i z e d s e p a r a t e l y , l y o p h i l i z e d a n d the s o l u b l e p r o t e i n s u b j e c t e d to one ( 1 - D ) o r t w o (2 -D) d i m e n s i o n a l e l e c t r o p h o r e s i s , o r , a l t e r n a t i v e l y , the m u s c l e s w e r e c u t i n h a l f a n d i m m e d i a t e l y f r o z e n . O n e h a l f was p r o c e s s e d f o r I C C a n d the o the r f o r m y o f i b r i l l a r A T P a s e h i s t o c h e m i s t r y . 60 METHODS IEF EDL and SOL REMOVED 2-D ELECTROPHORESIS 1 RIA IMMUNOBLOT 61 O ' F a r r e l l (1975) . T h e p r o t e i n b a n d t h o u g h t to be p a r v a l b u m i n i n the 3 2 - w e e k samp les was i d e n t i f i e d i n a ser ies o f c o m i g r a t i o n s tud ies w i t h a u t h e n t i c rat p a r v a l b u m i n . T h i s w o r k was r e p o r t e d p r e v i o u s l y b y J a s c h et a l . , 1982 a n d J a s c h a n d M o a s e (1985) a n d t h e r e f o r e , the t w o - d i m e n s i o n a l e l e c t r o p h o r e s i s d a t a o f the 3 2 - w e e k s a m p l e s was no t r e p e a t e d i n the p resen t s t u d y . i ) T i s s u e P r e p a r a t i o n . N o r m a l a n d d y s t r o p h i c m i c e w e r e k i l l e d b y c e r v i c a l d i s l o c a t i o n , a n d the E D L a n d so leus m u s c l e s w e r e d i s s e c t e d ou t i n to to f r o m r i g h t a n d l e f t h i n d l i m b s . T h e m u s c l e s w e r e w e i g h e d a n d h o m o g e n i z e d sepa ra te l y w i t h a g l a s s - T e f l o n pes t le h o m o g e n i z e r ( T R I - R I ns t rumen ts ) f o r 5 m i n i n 2 m l o f 1 m M t r is b u f f e r at p H 7.4. A n a l i q u o t o f h o m o g e n a t e c o n t a i n i n g 5 m g o f m u s c l e w e t w e i g h t was p l a c e d i n p o l y p r o p y l e n e tubes c o v e r e d w i t h M i r a c l o t h ( C h i c o p e e M i l l s ) , f r o z e n i n l i q u i d n i t r o g e n , a n d t h e n l y o p h i l i z e d f o r 20 h r . T h e s e samp les w e r e r u n o n 3 x 125 m m i s o e l e c t r i c f o c u s i n g ( I E F ) t u b e gels that w e r e s u b s e q u e n t l y used f o r t w o - d i m e n s i o n a l e l e c t r o p h o r e s i s . A l i q u o t s o f h o m o g e n a t e c o n t a i n i n g 11 m g o f m u s c l e w e t w e i g h t w e r e p r o c e s s e d w i t h the 5 m g a l i q u o t s , a n d s u b s e q u e n t l y r u n o n 5 x 125 m m I E F tube ge ls . T h e c o n c e n t r a t i o n o f the s a m p l e used f o r the I E F tube gels was based o n the w e i g h t / v o l u m e ra t i o o f 135 m g m u s c l e w e t w e i g h t / m l l y s i s b u f f e r d e s c r i b e d p r e v i o u s l y ( O ' F a r r e l l , 1975 ; J a s c h et a l . , 1982) . i i ) T w o - D i m e n s i o n a l E l e c t r o p h o r e s i s . L y o p h i l i z e d m u s c l e samp les f r o m the 2 - w e e k - o l d m i c e w e r e p a r t i a l l y s o l u b i l i z e d i n the l ys i s b u f f e r o f O ' F a r r e l l (1975) u s i n g p H 5 - 7 a m p h o l i n e s ( L K B ) . T h e s a m p l e was a p p l i e d to the t op o f e i t h e r 3 x 125 m m o r 5 x 125 m m I E F tube ge ls . T h e f i r s t d i m e n s i o n was r u n a c c o r d i n g to the m e t h o d o f O ' F a r r e l l ( 1975 ) , s e p a r a t i n g p a r v a l b u m i n f r o m the o t h e r m u s c l e p r o t e i n s b y t he i r i s o e l e c t r i c p o i n t s . T h e samp les w e r e f o c u s e d f o r 17 h r at 400 V a n d t h e n f o r 1 h r at 800 V . T h e 3 x 125 m m gels w e r e r e m o v e d f r o m the glass tubes b y e x p u l s i o n w i t h a i r t h r o u g h a 30 m l s y r i n g e . T h e s e gels w e r e s to red at - 7 0 ° C i n 16x150 m m c a p p e d c u l t u r e tubes c o n t a i n i n g 10 m l S D S s a m p l e b u f f e r ( 1 0 % g l y c e r o l , 5 % m e r c a p t o e t h a n o l , 2 . 3 % S D S , 62 .5 m M T r i s ) at p H 6.8 u n t i l r u n n i n g o f the s e c o n d d i m e n s i o n . T h e 5 x 62 125 m m gels w e r e r e m o v e d f r o m the glass tubes i n the same m a n n e r as w i t h the 3 x 125 m m ge l s , bu t t h e y w e r e t h e n p l a c e d i n t o 7 m m d i a m e t e r g lass tubes a n d f i x e d f o r 2 h r at r o o m t e m p e r a t u r e i n a s o l u t i o n c o n t a i n i n g 150 m l m e t h a n o l , 350 m l d i s t i l l e d w a t e r , 17.25 g m s u l f o s a l i c y l i c a c i d a n d 57.5 g m t r i c h l o r a c e t i c a c i d . T h e gels w e r e t h e n d e s t a i n e d f o r 30 m i n i n 2 5 % e t h a n o l a n d 8 % a c e t i c a c i d b e f o r e b e i n g s t a i n e d w i t h C o o m a s s i e B r i l l i a n t B l u e R 2 5 0 ( E a s t m a n , 0 .0287 g m / 5 0 m l d e s t a i n s o l u t i o n ) f o r 30 m i n i n a 6 0 ° C w a t e r b a t h . T h e gels w e r e t h e n r i n s e d w i t h d e s t a i n s o l u t i o n a n d s to red i n c a p p e d c u l t u r e tubes f o r 3 d a y s i n the d a r k , w i t h one to t w o s o l u t i o n c h a n g e s . T h e tube gels w e r e p h o t o g r a p h e d o n a f l u o r e s c e n t l i gh t b o x w i t h T e c h n i c a l P a n 2415 K o d a k f i l m . C o m i g r a t i o n o f a u t h e n t i c rat m u s c l e p a r v a l b u m i n w i t h the E D L h o m o g e n a t e samp les o n I E F a n d t w o - d i m e n s i o n a l gels was c o n d u c t e d i n a p r e v i o u s s t u d y ( Jasch a n d M o a s e , 1985) . C o m i g r a t i o n o f p u r e rat m u s c l e p a r v a l b u m i n w i t h the d y s t r o p h i c so leus h o m o g e n a t e o n 5 x 125 m m I E F gels was p e r f o r m e d i n one e x p e r i m e n t i n the p resen t s t u d y , as d e s c r i b e d p r e v i o u s l y b y J a s c h a n d M o a s e (1985) . T h e s e c o n d d i m e n s i o n was p e r f o r m e d o n 5 - 2 2 . 5 % e x p o n e n t i a l a c r y l a m i d e g r a d i e n t S D S s lab ge ls , 17 c m x 10 c m x 1 m m . T h e 3 x 125 m m I E F tube g e l was a d h e r e d to the t op o f the S D S s lab ge l w i t h 2 % aga rose , i n S D S s a m p l e b u f f e r , s u c h that the a c i d i c e n d o f the t u b e ge l c o u l d m i g r a t e d o w n the r i g h t - h a n d s i de o f the s lab g e l . A n o t c h was m a d e i n the agarose f o r m o l e c u l a r w e i g h t m a r k e r s ( B - g a l a c t o s i d a s e , 13 k i l o d a l t o n s ( K D ) ; p h o s p h o r y l a s e - a , 10 K D ; h u m a n s e r u m a l b u m i n , 66 K D ; p y r u v a t e k i n a s e , 57 K D ; D N A s e I, 43 K D ; s o y b e a n t r y p s i n i n h i b i t o r , 21 .5 K D ; l y s o z y m e , 14.3 K D ) n e x t to the a c i d i c e n d o f the g e l . A l l w e r e p u r c h a s e d f r o m M i l l i p o r e w h e r e a s b o v i n e s e r u m a l b u m i n (66 K D ) was o b t a i n e d f r o m S i g m a . E l e c t r o p h o r e s i s was c a r r i e d ou t as d e s c r i b e d b y O ' F a r r e l l (1975) f o r 1 0 - 1 2 h r at an i n i t i a l cons tan t c u r r e n t o f 25 m A . O n c e the d y e f r o n t h a d m i g r a t e d t h r o u g h the s t a c k i n g g e l , a b o u t 2 h r , the a m p e r a g e was i n c r e a s e d to 30 m A f o r the r e m a i n d e r o f the r u n . T h e s lab gels w e r e s o a k e d i n a d e a m p h o l i z i n g s o l u t i o n o f 5 0 % e t h a n o l , 7% ace t i c a c i d a n d 0 . 0 0 5 % C o o m a s s i e B l u e R 2 5 0 63 f o r 36 h r . A f t e r the gels w e r e r e h y d r a t e d f o r 2 h r i n 7% a c e t i c a c i d a n d 0 . 0 0 5 % C o o m a s s i e B l u e R 2 5 0 , the p r o t e i n a n d p o l y p e p t i d e spots w e r e v i s u a l i z e d b y s t a i n i n g f o r 30 m i n i n 5 0 % t r i c h l o r o a c e t i c a c i d ( T C A ) a n d 0 . 1 % C o o m a s s i e B l u e R 2 5 0 . T h e gels w e r e d e s t a i n e d f o r s e v e r a l d a y s i n 7% a c e t i c a c i d , v a c u u m sea led i n p l a s t i c bags , a n d p h o t o g r a p h e d o n a l i g h t b o x w i t h T e c h n i c a l P a n 2415 K o d a k f i l m . A n t i s e r a p r o d u c t i o n P r i o r to the i n i t i a l i m m u n i z a t i o n i n j e c t i o n s , a p p r o x i m a t e l y 50 m l o f p r e i m m u n e s e r u m was c o l l e c t e d f r o m rabb i t s a n d 100 u l a l i quo t s w e r e s to red f o r use la ter as c o n t r o l a n t i s e r u m . A n t i s e r a to m o u s e a n d rat s ke le ta l m u s c l e p a r v a l b u m i n was ra i sed i n 2 a d u l t r a b b i t s b a s e d o n the p r o c e d u r e d e s c r i b e d b y L a z a r i d e s a n d H u b b a r d (1976) . R a t p a r v a l b u m i n p u r i f i e d b y h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y ( H P L C ) was p u r c h a s e d f r o m C a l B i o c h e m - B e h r i n g . M o u s e m u s c l e p a r v a l b u m i n was r e s o l v e d f r o m c r u d e m u s c l e h o m o g e n a t e samp les o b t a i n e d f r o m the m e d i a l g a s t r o c n e m i u s o f n o r m a l a d u l t m i c e . P a r v a l b u m i n was r e s o l v e d i n 5 m m x 125 m m i s o e l e c t r i c f o c u s i n g ( I E F ) ge l s , as d e s c r i b e d a b o v e , as a s i ng l e p r o m i n e n t b a n d at p i = 5 .45 . A n u m b e r o f these bands w e r e c a r e f u l l y e x c i s e d f r o m the s t a i n e d ge l s , a n d e m u l s i f i e d i n 1 m l 0.15 M p h o s p h a t e b u f f e r at p H 7.4 a n d 1 m l F r e u n d ' s c o m p l e t e a d j u v a n t ( D I F C O L a b o r a t o r i e s ) . T h e e m u l s i o n was s u b s e q u e n t l y i n j e c t e d i n t r a m u s c u l a r i l y i n t o the h i n d l i m b o f e a c h r a b b i t , a l t e r n a t i n g r i g h t a n d l e f t s ides w i t h e a c h boos te r i n j e c t i o n . E a c h i n j e c t i o n c o n t a i n e d a p p r o x i m a t e l y 1 5 0 - 2 0 0 p g p a r v a l b u m i n . T h e a m o u n t o f p a r v a l b u m i n i n e a c h b a n d was e s t i m a t e d b y s i m u l t a n e o u s l y r u n n i n g 50 p g q u a n t i t i e s o f the p u r e rat p a r v a l b u m i n o n I E F gels as a s t a n d a r d f o r s p e c t r o p h o t o m e t r i c r e a d i n g s . G e l s w e r e s c a n n e d o n a G i l f o r d s p e c t r o p h o t o m e t e r ( L e e d s a n d N o r t h r u p ) b e f o r e the b a n d s w e r e r e m o v e d , a n d the a b s o r b a n c e was c o m p a r e d to the rat p a r v a l b u m i n s t a n d a r d . I m m u n i z a t i o n s w e r e i n 2 - w e e k i n t e r v a l s . T h e f i r s t b l e e d i n g was p e r f o r m e d 10 d a y s a f t e r the e i g h t h w e e k . A p p r o x i m a t e l y 1 m l o f b l o o d was c o l l e c t e d i n t o a n e q u a l v o l u m e o f p h o s p h a t e b u f f e r e d sa l i ne ( P B S ) , 5 m M e t h y l e n e g l y c o l - b i s - B - a m i n o e t h y l e the r 64 ( E G T A ) a n d 0 . 0 2 % s o d i u m a z i d e ( p H 7.3). T h e b l o o d was c e n t r i f u g e d f o r 15 m i n at 2 5 0 0 x g , a n d a l i quo t s o f the s e r u m w e r e s to red at - 7 0 ° C . S p e c i f i c i t y o f the an t i se ra was tes ted b y the p r o c e d u r e s d e s c r i b e d b e l o w . T h e r a b b i t i n w h i c h the a n t i s e r a to m o u s e p a r v a l b u m i n was r a i s e d was b l e d a to ta l o f 3 t imes o v e r a 52 - w e e k p e r i o d , w h e r e a s the r a b b i t i n w h i c h the a n t i s e r a to rat p a r v a l b u m i n was r a i s e d was b l e d a to ta l o f 5 t imes o v e r 78 w e e k s . E a c h b l e e d i n g resu l t ed i n a p p r o x i m a t e l y 30 m l o f a n t i s e r u m . T h e r a b b i t s w e r e b l e d b y a s m a l l i n c i s i o n to the m a r g i n a l v e i n o f the r i g h t ea r , a n d the b l o o d was a l l o w e d to d r i p i n to a g lass b e a k e r c o n t a i n i n g P B S , E G T A a n d s o d i u m a z i d e . T h e b l o o d was c e n t r i f u g e d f o r 30 m i n at 5000 x g a n d the s e r u m was p o o l e d . Separa te a l i q u o t s o f 20 m l , 1.5 m l a n d 100 u l w e r e s to red e i t h e r at - 7 0 ° C o r at - 2 0 ° C . C h a r a c t e r i z a t i o n o f the A n t i b o d y T h e a n t i b o d y to m o u s e s k e l e t a l m u s c l e P V was c h a r a c t e r i z e d b y the f o l l o w i n g m e t h o d s . T r i p l i c a t e e n z y m e - l i n k e d i m m u n o s o r b e n t assays ( E L I S A ) w e r e c o n d u c t e d to d e t e r m i n e the t i t re o f the a n t i s e r u m a n d i ts c r o s s - r e a c t i v i t y w i t h p u r i f i e d rat s ke le ta l m u s c l e P V . M o n o s p e c i f i c i t y o f the a n t i s e r u m was e v a l u a t e d b y p o l y a c r y l a m i d e ge l e l e c t r o p h o r e s i s ( P A G E ) a n d W e s t e r n b l o t t i n g , a n d r a d i o i m m u n o a s s a y ( R I A ) . i ) E n z y m e - L i n k e d I m m u n o s o r b a n t A s s a y ( E L I S A ) . T h e s t a n d a r d E L I S A test u s e d i n th is s t u d y was based o n that d e s c r i b e d p r e v i o u s l y ( E n g v a l l a n d P e r l m a n n , 1971 ; B u t l e r et a l . , 1978; K e l l y et a l . , 1979) . F a l c o n 3912 m i c r o t e s t assay p la tes ( F i s h e r ) w e r e c o a t e d w i t h p u r e rat p a r v a l b u m i n ( C a l B i o c h e m ) at c o n c e n t r a t i o n s o f 2 5 , 5 0 , 75 a n d 100 n g / w e l l d i s s o l v e d i n c a r b o n a t e b u f f e r ( p H 9 .6 ) , a n d i n c u b a t e d o v e r n i g h t at 4 ° C . T h e p la tes w e r e w a s h e d th ree t i m e s w i t h P B S - T w e e n , a n d the s e r u m to be tes ted f o r a n t i - m o u s e p a r v a l b u m i n was t h e n a d d e d i n d o u b l i n g d i l u t i o n s o f 1:25 to 1:3200. F o l l o w i n g a n i n c u b a t i o n f o r 90 m i n at r o o m t e m p e r a t u r e w i t h s u b s e q u e n t w a s h i n g , the d e v e l o p i n g s e c o n d a n t i b o d y , sheep a n t i - r a b b i t i m m u n o g l o b u l i n c o n j u g a t e d to a l k a l i n e phospha tase ( S A R I g ) was a p p l i e d at a 1:1000 d i l u t i o n . A f t e r a f i n a l 90 m i n i n c u b a t i o n a n d b u f f e r 65 w a s h , p - n i t r o p h e n y l p h o s p h a t e d i s o d i u m ( S i g m a , 5 m g / 5 m l 10% d i e t h a n o l a m i n e b u f f e r ) , was a d d e d a n d the r e a c t i o n was a l l o w e d to c o n t i n u e f o r 30 m i n . T h e r e a c t i o n was m e a s u r e d f o r a b s o r b a n c e at 4 0 5 n m o n a D y n a t e c h M R 580 m i c r o e l i s a r eade r . E a c h test was r u n i n t r i p l i c a t e , a n d the p r e i m m u n e r a b b i t s e r u m was u s e d as a c o n t r o l f o r e a c h d i l u t i o n . T h e resu l ts ( F i g . 19) i n d i c a t e d that the t i te r o f the a n t i s e r u m aga ins t m o u s e p a r v a l b u m i n was i n the range o f 1:200 - 1:1600. A w o r k i n g d i l u t i o n o f 1:400 was c h o s e n f o r a l l i m m u n o h i s t o c h e m i s t r y e x p e r i m e n t s . i i ) T i s s u e P r e p a r a t i o n f o r I m m u n o c h e m i c a l S t u d i e s . P r e p a r a t i o n o f t issue ex t rac ts tha t w e r e u s e d f o r o n e - d i m e n s i o n a l ge l e l e c t r o p h o r e s i s , i m m u n o b l o t s , a n d r a d i o i m m u n o a s s a y s was as f o l l o w s . I n d i v i d u a l m u s c l e s w e r e w e i g h e d a n d m i n c e d w i t h a r a z o r b l a d e . T w o to f o u r o f the m i n c e d a d u l t m u s c l e s w e r e c o m b i n e d a n d h o m o g e n i z e d i n 20 t i m e s the v o l u m e o f c o l d T r i s b u f f e r (62.5 m M T r i s , 1 m M E D T A , a n d 0 . 0 2 % s o d i u m a z i d e ) at p H 6.7 w i t h a T e k m a r T i s s u m i z e r h o m o g e n i z e r . T h e n o r m a l 2 - w e e k m u s c l e s w e r e c o m b i n e d to g i v e a n a p p r o x i m a t e f i n a l w e t w e i g h t o f 10 m g w h i c h was a lso h o m o g e n i z e d i n 20 t imes the v o l u m e o f T r i s b u f f e r . D u e to the s c a r c i t y o f h o m o z y g o u s d y s t r o p h i c neona tes , the 2 - w e e k d y s t r o p h i c m u s c l e s w e r e c o m b i n e d to g i v e a n a p p r o x i m a t e f i n a l w e t w e i g h t o f 5 m g w h i c h was h o m o g e n i z e d i n 500 u l T r i s b u f f e r at 5 0 % the c o n c e n t r a t i o n (31.25 m M T r i s , 0.5 m M E D T A a n d 0 . 0 1 % s o d i u m a z i d e ) . A l l h o m o g e n a t e s w e r e c e n t r i f u g e d at 39 ,000 x g f o r 45 m i n at 4 ° C , a n d the s u p e r n a t a n t was d i v i d e d i n t o 100 u l a l i q u o t s . M u s c l e ex t rac t s used f o r ge l e l e c t r o p h o r e s i s a n d i m m u n o b l o t t i n g w e r e d i v i d e d i n t o a l i q u o t s i n 0.5 m l E p p e n d o r f v i a l s , a n d l y o p h i l i z e d f o r 1 2 - 1 8 h r . T h e s e f r e e z e - d r i e d samp les w e r e s t o r e d at - 2 0 ° C u n t i l the S D S s lab gels w e r e r u n . T h e s u p e r n a t a n t u s e d f o r the R I A was s to red i n 10x12 c m s i l i c o n i z e d glass tubes at - 2 0 ° C u n t i l the assay was p e r f o r m e d . i i i ) I m m u n o b l o t s . E l e c t r o p h o r e t i c b l o t t i n g o f p r o t e i n s was p e r f o r m e d a c c o r d i n g to the p r o c e d u r e o f T o w b i n et a l . , (1979) . T h e samp les u s e d i n these e x p e r i m e n t s w e r e a l i quo t s o f the m u s c l e s o r i g i n a l l y h o m o g e n i z e d i n t w e n t y t imes the v o l u m e t r is s a m p l e b u f f e r as d e s c r i b e d p r e v i o u s l y . T h e lOOp l f r e e z e - d r i e d t issue ex t rac t s f r o m n o r m a l a n d 66 F i g u r e 19 B i n d i n g response o f the a n t i - m o u s e p a r v a l b u m i n a n t i s e r u m at v a r i o u s d i l u t i o n s , to d e c r e a s i n g c o n c e n t r a t i o n s o f the rat m u s c l e p a r v a l b u m i n ( a n t i g e n c o n c e n t r a t i o n ) , as d e t e c t e d b y a n e n z y m e - l i n k e d i m m u n o s o r b a n t assay ( E L I S A ) . A b s o r b a n c e was m e a s u r e d at 405 n m . A n t i g e n c o n c e n t r a t i o n s w e r e m e a s u r e d i n n g / l O O u l . T h e va lues g i v e n are the m e a n s o f t r i p l i c a t e assays , n o r m a l i z e d at e a c h d i l u t i o n to the b a c k g r o u n d n o n - s p e c i f i c b i n d i n g (abso rbance o f 0 .065) d e t e r m i n e d b y the p r e i m m u n e c o n t r o l s e r u m . 67  d y s t r o p h i c m i c e at 32 a n d 2 w e e k s o f age , w e r e r e c o n s t i t u t e d w i t h 50 p i o f 10% g l y c e r o l , 2 % S D S , a n d 0.1 M d i t h i o t h r e i t o l ( D T T ) . T h e y w e r e s u b s e q u e n t l y a p p l i e d i n d u p l i c a t e ha l ves to a 5 - 2 2 . 5 % e x p o n e n t i a l g r a d i e n t S D S s lab g e l , a n d r u n a c c o r d i n g to the s e c o n d d i m e n s i o n o f the t w o - d i m e n s i o n a l t e c h n i q u e d e s c r i b e d a b o v e . F o r a c o n t r o l , 25 p g o f a u t h e n t i c rat m u s c l e p a r v a l b u m i n was r u n o n the h a l f o f the g e l to be b l o t t e d . A t the e n d o f the r u n the s lab g e l was d i v i d e d i n h a l f , a n d one h a l f was t h e n s t a i n e d a n d s u b s e q u e n t l y d e s t a i n e d as d e s c r i b e d a b o v e . T h e c o r r e s p o n d i n g h a l f was n o t c h e d i n one o f the l o w e r c o r n e r s a n d e q u i l i b r a t e d i n 500 m l o f t r a n s f e r b u f f e r (25 m M T r i s , 192 m M g l y c i n e , 2 0 % v / v m e t h a n o l ) f o r 30 m i n at r o o m t e m p e r a t u r e . T h e p r o t e i n s w e r e t h e n e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d to a 0.45 p m p o r e n i t r o c e l l u l o s e m e m b r a n e o n a B i o - R a d T r a n s - B l o t C e l l a n d p o w e r s u p p l y ( M o d e l 2 5 0 / 2 . 5 ) . T h e t r a n s f e r was r u n e i t h e r c o o l e d f o r 3 h r at h i g h i n t e n s i t y (150 V ) o r c o o l e d o v e r n i g h t at 40 V . A f t e r the n i t r o c e l l u l o s e m e m b r a n e was d r i e d f o r 30 m i n at r o o m t e m p e r a t u r e , i t was w a s h e d f o r 1 h r i n a b l o c k i n g s o l u t i o n o f 20 m M T r i s , 500 m M N a C l , 3 % f e t a l c a l f s e r u m ( F C S , C a l B i o c h e m - B e h r i n g ) , . a n d 1% N G S p H 7 .5 , to satura te the f r e e b i n d i n g - s i t e s . T h e ge l was s t a i n e d a n d d e s t a i n e d as d e s c r i b e d a b o v e , a n d e x a m i n e d c a r e f u l l y f o r a n y r e m a i n i n g p r o t e i n . T h e m e m b r a n e was f u r t h e r w a s h e d i n T r i s - b u f f e r e d sa l i ne c o n t a i n i n g 0 . 0 5 % T w e e n - 2 0 ( T T B S , B i o - R a d ) f o r 5 m i n a n d t h e n i n c u b a t e d i n a n t i - m o u s e p a r v a l b u m i n a n t i s e r u m (1:200) o v e r n i g h t at 4 ° C . F o l l o w i n g t w o 5 m i n washes i n T T B S , the m e m b r a n e was i n c u b a t e d i n goat a n t i - r a b b i t I g G - h o r s e r a d i s h p e r o x i d a s e c o n j u g a t e (1 :3000 , B i o - R a d ) at r o o m t e m p e r a t u r e f o r 2 h r . T h e m e m b r a n e was a g a i n w a s h e d t w i c e i n T T B S a n d t h e n f o r a f i n a l 5 m i n i n T B S . C o l o u r d e v e l o p m e n t was a c h i e v e d b y s o a k i n g the b lo ts i n a 100 m l T B S s o l u t i o n c o n t a i n i n g 0 . 0 1 5 % H 2 0 2 a n d 60 m g 4 - c h l o r o - l - n a p t h o l ( S i g m a ) w h i c h was f i r s t d i s s o l v e d i n 20 m l c o l d m e t h a n o l . A l l i n c u b a t i o n s a n d washes w e r e d o n e w i t h gen t l e a g i t a t i o n . T h e r e a c t i o n was t e r m i n a t e d a f t e r 2 0 - 3 0 m i n b y repea ted w a s h i n g i n d i s t i l l e d w a t e r , a n d the gels w e r e p h o t o g r a p h e d w h i l e w e t w i t h T e c h n i c a l P a n 2415 K o d a k f i l m . T h e b lo ts w e r e s to red a n d p r o t e c t e d f r o m l i g h t , o n f i l t e r p a p e r c o v e r e d w i t h p l as t i c w r a p . 69 The spots from one of the 2-week normal EDL two-dimensional runs were cut out of the gel, and the proteins were eluted separately in 300 ul of 0.1% SDS at 4°C for 2-3 days. They were then minced and mashed, and centrifuged at 4,000 x g to separate the gel fragments. A 200 pi volume of 125 mM borate buffer (25 mM sodium borate, 75 mM NaCl, 1 mM EDTA, pH 8.4 ) was added to the supernatant and the PV concentration determined by RIA, as described below. Only the extracts from the spot believed to be parvalbumin resulted in complete displacement of 1 2 5 I -PV binding to the antiserum used in the RIA. All the other spot extracts showed no displacement indicating an absence of parvalbumin. iv) Radioimmunoassay. Cross-reactivity of the anti-mouse PV antiserum was tested on a RIA against two other calcium-binding proteins, bovine brain calmodulin and calcium-binding protein (CaBP). Calmodulin (16,723 daltons) was purchased from Calbiochem whereas CaBP was purified by Dr. Baimbridge, of the Department of Physiology, from bovine cerebellum. The principle for the RIA used in this study is outlined in Figure 20 and the general procedure for all the radioimmunoassays used in this study is as follows. Rat skeletal muscle parvalbumin (CalBiochem) was iodinated according to the Chloramine-T technique of Greenwood and Hunter (1963). Since mouse or rat PV contains no tyrosine residues (Table II) the pH of the iodination buffer was increased from 7.4 to 8.6 in order to tag histidine. Briefly, 5 pg of rat parvalbumin was dissolved in 10 pi of 0.05 M disodium hydrogen phosphate buffer at pH 8.6, 10 pi of 1 2 5 I (Amersham), and 10 pi of Chloramine-T (5 mg/ml buffer). This solution was kept on ice and the reaction was allowed to proceed for 2 min. Iodination was stopped by the addition of 10 pi of Na metabisulphite (24 mg/ml buffer) and quenched with 1 ml potassium iodine (10 mg/ml buffer). Unreacted iodine was separated from that bound to the protein on a Sephadex G-25 column, and eluted with 0.05 M sodium phosphate buffer (pH 7.4), 2 g/1 BSA and 0.02% sodium azide at 4°C. The resulting 1 2 5 I - parvalbumin had a specific radioactivity of 3 Ci/m mole. 70 Figure 20 Schematic diagram outlining the radioimmunoassay procedure used to quantitate the parvalbumin content in muscle samples. Unlabelled antigen (Ag), parvalbumin, in the muscle sample is mixed with a known dilution of antibody (Ab), so that the system is in antigen excess. Labelled parvalbumin (125I) of a known concentration is added to the solution, and also becomes Ag-bound. The amount of labelled parvalbumin bound to the antibody is inversely proportional to non-labelled parvalbumin in the muscle sample. Free parvalbumin is separated from the bound 1 2 5 I parvalbumin by an excess of gamma-globulin, which precipitates the labelled complex. Radioactivity in the immunoprecipitant is counted on a gamma counter and the percent-binding is plotted as a function of the amount of competition added. 71 R I A cm Antigen & Antibody in fafo muscle sample Incubat ion -"O Add Radioactive Label ( 1 2 5l) -> -D3 fe fe fe Incubation Separate bound and free fraction with 2°Ab Count radioactivity bound to ^4 ^4 immunoprecipitate 7 Z A l l assays w e r e p e r f o r m e d i n t r i p l i c a t e i n 10x75 c m glass tubes k e p t o n i c e . A l l d i l u t i o n s w e r e m a d e i n the R I A b u f f e r p r e p a r e d as f o l l o w s . T h e s tock b a r b i t a l b u f f e r c o n t a i n e d 8 g o f b a r b i t u r i c a c i d d i s s o l v e d i n 1000 m l o f d i s t i l l e d w a t e r , w h i l e h e a t i n g , to w h i c h 41 .2 g o f s o d i u m b a r b i t a l w e r e a d d e d a n d the v o l u m e was b r o u g h t to 2 0 0 0 m l w i t h d i s t i l l e d w a t e r ( p H 8 . 2 - 8 . 5 ) . T h e w o r k i n g b u f f e r was t h e n p r e p a r e d b y m i x i n g 2 5 0 m l o f s tock b a r b i t a l b u f f e r w i t h 6 g / L B S A , 1 m M E D T A a n d 0 . 0 2 % s o d i u m a z i d e . T h e r e a c t i o n m i x t u r e c o n t a i n e d 700 p i o f b u f f e r , 100 p i o f a n t i s e r u m (1:400) , a n d 100 p i o f e i t h e r s t a n d a r d o r s a m p l e . T h e d i l u t i o n ser ies u s e d f o r the P V s tanda rd (rat s k e l e t a l m u s c l e f r o m C a l b i o c h e m ) r a n g e d f r o m 80 n g / l O O p l to 0 .156 n g / l O O p l . C a B P was u s e d i n a d i l u t i o n ser ies o f 240 to 7.5 n g / 1 0 0 p i . C a l m o d u l i n was used i n d i l u t i o n s o f 1 p g / 1 0 0 p i to 31 .25 n g / 1 0 0 p i . M a x i m u m b i n d i n g was m e a s u r e d i n tubes c o n t a i n i n g 800 p i o f b u f f e r a n d 100 p i o f e i t h e r s t a n d a r d o r s a m p l e . T u b e s f o r n o n s p e c i f i c b i n d i n g ( N S B ) measu res c o n t a i n e d o n l y b u f f e r . T h e assays w e r e i n c u b a t e d o v e r n i g h t at 4 °C f o l l o w e d b y a d d i t i o n o f 100 p i o f l a b e l l e d p a r v a l b u m i n (10 ,000 c p m / t u b e ) . T h e assay was t h e n r e t u r n e d to the c o l d ( 4 °C ) f o r 24 h r . F o r the R I A d e s i g n e d to test c r o s s - r e a c t i v i t y o f the a n t i s e r u m the s tanda rds a n d l a b e l w e r e a d d e d o n the same d a y a n d i n c u b a t e d o v e r n i g h t at 4 °C . B o u n d a n d f r e e f r a c t i o n s w e r e sepa ra ted b y the a d d i t i o n o f 100 p i o f b o v i n e g a m m a g l o b u l i n (8 m g / m l sa l i ne ) a n d 1 m l o f P E G ( 2 0 % i n d H 2 0 ) . T h e assay tubes w e r e v o r t e x m i x e d u n t i l t h e y a p p e a r e d c l o u d y a n d t h e n c e n t r i f u g e d at 4 0 0 0 x g a n d 4°C f o r 30 m i n . T h e s u p e r n a t a n t was d r a i n e d b y i n v e r s i o n o f the tubes o v e r a s i n k . T h e tubes w e r e w a s h e d g e n t l y w i t h a s t r e a m o f tap w a t e r w h i l e i n v e r t e d a n d a l l o w e d to d r y p r i o r to d e t e c t i o n o f 1 2 5 I o n a B e c k m a n m o d e l 1180 g a m m a c o u n t e r . S t a n d a r d c u r v e s s h o w i n g the r e l a t i o n s h i p b e t w e e n p e r c e n t b i n d i n g a n d the a m o u n t o f u n l a b e l l e d p a r v a l b u m i n w e r e p l o t t e d o n s e m i - l o g a r i t h m i c p a p e r . P e r c e n t b i n d i n g was c a l c u l a t e d as: c o u n t s ( s tanda rd o r s a m p l e ) - N S B x 100 to ta l c o u n t s 73 S P E C I F I C M E T H O D S I m m u n o h i s t o c h e m i s t r v N o r m a l (+/+) a n d d y s t r o p h i c ( d y 2 J / d y 2 J ) m i c e at 2 a n d 32 w e e k s o f age w e r e k i l l e d b y c e r v i c a l d i s l o c a t i o n . T h e 2 w e e k d y s t r o p h i c a n i m a l s w e r e o b t a i n e d b y the a m p u t a t i o n p r o c e d u r e d e s c r i b e d a b o v e . In an a t t emp t to c o r r e l a t e m u s c l e f i b e r - t y p e a n d p a r v a l b u m i n c o n t e n t , the f a s t - t w i t c h e x t e n s o r d i g i t o r u m l o n g u s ( E D L ) a n d s l o w - t w i t c h so leus m u s c l e s w e r e d i s s e c t e d ou t , cu t t h r o u g h the m i d - b e l l y r e g i o n s a n d e a c h h a l f was p r o c e s s e d sepa ra te l y e i t he r f o r i m m u n o h i s t o c h e m i c a l o r h i s t o c h e m i c a l s t a i n i n g p r o c e d u r e s . D u e to the h i g h s o l u b i l i t y o f p a r v a l b u m i n , the use o f u n f i x e d o r p o s t f i x e d c r yos ta t sec t i ons was u n s u i t a b l e f o r i m m u n o h i s t o c h e m i s t r y . M a n y f i x a t i o n a n d e m b e d d i n g p r o c e d u r e s w e r e i n i t i a l l y i n v e s t i g a t e d as o u t l i n e d i n T a b l e I V . S o m e o f the t issue was e m b e d d e d i n E p o n , a n d a l te rna te s e m i - t h i c k (1 p m ) a n d t h i n ( g o l d to s i l v e r ) p l a s t i c - s e c t i o n s w e r e s t a i n e d f o r P V i n an a t t emp t to co r re l a te the u l t r a s t r u c t u r a l a n d i m m u n o h i s t o c h e m i c a l l o c a l i z a t i o n o f th is p r o t e i n . P e n e t r a t i o n o f the a n t i b o d y i n the s e m i - t h i c k p l a s t i c - s e c t i o n s was p o o r a n d the s t a i n i n g was u n r e l i a b l e , so that th is m e t h o d was no t p u r s u e d a n y f u r t h e r . T h e mos t p r e c i s e l o c a l i z a t i o n s o f p a r v a l b u m i n w e r e a c h i e v e d w i t h a s o m e w h a t c u m b e r s o m e a n d l e n g t h y f r e e z e - s u b s t i t u t i o n f i x a t i o n p r o t o c o l ( F e b e r a n d S i d m a n , 1958; P e a r s e , 1968 , 1980) f o l l o w e d b y p a r a f f i n e m b e d d i n g . A p r e l i m i n a r y test o f the a n t i s e r a t i te r ( d i l u t i o n range f r o m 1:10 to 1:3200) was c o n d u c t e d o n m o u s e c e r e b e l l u m w h i c h is k n o w n to be p o s i t i v e f o r p a r v a l b u m i n ( C e l i o a n d H e i z m a n n , 1981) . A su i t ab le a n t i s e r a c o n c e n t r a t i o n was c h o s e n b a s e d o n th is s c r e e n i n g a n d o n the E L I S A test. T h e i m m u n o h i s t o c h e m i c a l m e t h o d used was a m o d i f i c a t i o n o f the u n l a b e l e d a n t i b o d y e n z y m e - l i n k e d t e c h n i q u e o f S t e r n b e r g e r (1979) . M u s c l e ha l ves w e r e f r o z e n i n i sopen tane c o o l e d b y l i q u i d n i t r o g e n a n d t h e n i m m e d i a t e l y i m m e r s e d i n t o a v i a l o f 1% p i c r i c / e t h a n o l f i x a t i v e k e p t at - 7 0 ° C . T h e v i a l s w e r e s u r r o u n d e d b y d r y i ce a n d t h e n s to red at - 7 0 ° C f o r 2 w e e k s . T h e t issue was s l o w l y d e h y d r a t e d f o r p a r a f f i n e m b e d d i n g a c c o r d i n g to the f o l l o w i n g steps: (a) 74 th ree c h a n g e s i n e t h a n o l o v e r 1 2 - 2 4 h r at 4 ° C , (b) i m m e r s i o n f o r 6 - 1 2 h r i n c h l o r o f o r m at 4 ° C , (c) i m m e r s i o n f o r 1 h r i n c h l o r o f o r m at r o o m t e m p e r a t u r e . P a r a f f i n sec t i ons (10 u m t h i c k ) w e r e c o l l e c t e d o n a l b u m i n - c o a t e d glass s l i d e s , a n d d r i e d e i t h e r f o r 2 h r at 6 0 ° C o r f o r 2 4 - 4 8 h r at 40 °C . A f t e r d e p a r a f f i n i z a t i o n a n d s u b s e q u e n t r e h y d r a t i o n i n a g r a d e d ser ies o f e t h a n o l , the sec t i ons w e r e b l o c k e d i n 10% H 2 0 2 f o r 15 m i n , w a s h e d f o r 30 m i n i n P B S , a n d t h e n i n c u b a t e d i n 5 % n o r m a l goat s e r u m ( N G S ) ( M i l e s ) f o r 30 m i n to r e d u c e n o n - s p e c i f i c s t a i n i n g . T h e a n t i s e r u m aga ins t m o u s e p a r v a l b u m i n was a p p l i e d to the sec t i ons at a 1:400 d i l u t i o n i n a n t i b o d y b u f f e r at p H 7.3 ( P B S , 0 . 3 % T r i t o n X - 1 0 0 , 3 % N G S ) a n d i n c u b a t e d o v e r n i g h t i n a m o i s t c h a m b e r at 4 ° C . A f t e r the sec t ions w e r e w a s h e d i n P B S f o r 15 m i n , t h e y w e r e i n c u b a t e d i n a m o i s t c h a m b e r at r o o m t e m p e r a t u r e f o r 90 m i n i n the s e c o n d a n t i b o d y , goat a n t i - r a b b i t ( G A R ) I g G (1 :100 , M i l e s ) . A f t e r a n o t h e r 15 m i n w a s h i n P B S , sec t ions w e r e i n c u b a t e d w i t h the r a b b i t p e r o x i d a s e a n t i - p e r o x i d a s e ( P A P ) c o m p l e x (1 :100 , C a p p e l ) f o r 90 m i n i n a m o i s t c h a m b e r at r o o m t e m p e r a t u r e . B o t h the G A R a n d P A P w e r e p r e p a r e d i n the a n t i b o d y b u f f e r c o n t a i n i n g n o r m a l goat s e r u m . F i n a l washes i n P B S w e r e f o l l o w e d b y a r i n s e i n 5 m M T r i s - H C l b u f f e r ( T B ) at p H 7.6. T h e e n z y m e r e a c t i o n was d e v e l o p e d w i t h d i a m i n o b e n z i d i n e / H 2 0 2 ( S i g m a ) , 30 m g / 1 0 0 m l P B S c o n t a i n i n g 100 p i 3 % H 2 0 2 , f o r abou t 10 m i n , a f t e r w h i c h the sec t i ons w e r e r i n s e d i n T B to s top the r e a c t i o n . T h e sec t i ons w e r e t h e n d e h y d r a t e d t h r o u g h a n a s c e n d i n g ser ies o f e t h a n o l , c l e a r e d i n x y l e n e , a n d c o v e r s l i p p e d w i t h C y t o s e a l 60 (S tephens S c i e n t i f i c ) . C o n t r o l s l i des w e r e p r o c e s s e d i d e n t i c a l l y a n d s i m u l t a n e o u s l y w i t h p r e i m m u n e s e r u m f r o m the same a n i m a l . A n t i s e r u m p r e a b s o r b e d o v e r n i g h t w i t h p u r i f i e d rat m u s c l e p a r v a l b u m i n was a lso u s e d as a c o n t r o l . Sec t i ons w e r e e x a m i n e d a n d p h o t o g r a p h e d o n a L e i t z O r t h o p l a n p h o t o m i c r o s c o p e u s i n g T e c h n i c a l P a n 2415 K o d a k f i l m . H i s t o c h e m i s t r y A m o d i f i e d m e t h o d f o r m y o s i n A T P a s e s t a i n i n g was a d o p t e d because o f i ts ease o f 75 a p p l i c a t i o n ( D o r i g u z z i et a l . , 1983) . T h i s m e t h o d has the a d v a n t a g e o f b e i n g less t i m e c o n s u m i n g t h a n p r e v i o u s l y p u b l i s h e d p r o t o c o l s . It a lso e l i m i n a t e s the use o f a m m o n i u m s u l f i d e , a n d a l l th ree e x t r a f u s a l f i b e r t ypes c o u l d be d i s t i n g u i s h e d s i m u l t a n e o u s l y w i t h th is p r o c e d u r e u s i n g the same p H . T h e r e s p e c t i v e ha l ves o f the E D L a n d so leus m u s c l e s , c o r r e s p o n d i n g to those p r o c e s s e d f o r i m m u n o h i s t o c h e m i s t r y , w e r e e m b e d d e d i n f r e s h m o u s e l i v e r a n d f r o z e n i n i s o p e n t a n e c o o l e d b y l i q u i d n i t r o g e n , a n d s to red at - 7 0 ° C u n t i l r e q u i r e d f o r s e c t i o n i n g . S e r i a l c r y o s t a t - s e c t i o n s (10 u m t h i c k ) w e r e c o l l e c t e d o n glass c o v e r s l i p s a n d a i r d r i e d to r o o m t e m p e r a t u r e b e f o r e s t a i n i n g . T h e sec t i ons w e r e s t a i n e d f o r m y o f i b r i l l a r c a l c i u m - A T P a s e at p H 4.6 a c c o r d i n g to the m e t h o d o f D o r i g u z z i (1983) . T h e c o v e r s l i p s w e r e p r e i n c u b a t e d at r o o m t e m p e r a t u r e f o r 5 - 6 m i n at p H 4 .6 , a n d t h e n w a s h e d t w i c e f o r 60 sec ( two c h a n g e s ) i n the bas i c m e d i u m . A f t e r a 20 m i n i n c u b a t i o n at 37°C i n the A T P b a s i c m e d i u m , the sec t i ons w e r e r i n s e d i n 1% t o l u i d i n e b l u e f o r 20 sec a n d t h e n r i n s e d w e l l w i t h d i s t i l l e d wa te r . C o v e r s l i p s w e r e t h e n r i n s e d i n a s c e n d i n g e t h a n o l ( 7 0 , 90 a n d 100%) f o r 2 m i n e a c h , c l e a r e d i n x y l e n e , a n d m o u n t e d i n C y t o s e a l 60 (S tephens S c i e n t i f i c ) . P h o t o g r a p h s w e r e t a k e n o n a L e i t z O r t h o p l a n p h o t o m i c r o s c o p e u s i n g T e c h n i c a l P a n 2415 K o d a k f i l m . S ta t i s t i ca l A n a l y s i s M u s c l e f i b e r s w e r e c a t e g o r i z e d a n d c o u n t e d a f t e r P V i m m u n o h i s t o c h e m i c a l a n d m y o s i n A T P a s e s t a i n i n g . T h e y w e r e d e s i g n a t e d l i g h t ( L ) , d a r k ( D ) , o r i n t e r m e d i a t e (I) f i b e r - t y p e s u s i n g a C a m e r a L u c i d a a t t a c h m e n t to a L e i t z b r i g h t f i e l d m i c r o s c o p e . In the case o f the m y o s i n A T P a s e s t a i n i n g , th is n o m e n c l a t u r e c o r r e s p o n d s to the m o r e c o m m o n l y u s e d c l a s s i f i c a t i o n s ( B r o o k a n d K a i s e r , 1970; P e t e r et a l . , 1972): D = t ype 1 / s l o w - t w i t c h o x i d a t i v e ( S O ) , L = t y p e 2 A / f a s t - t w i t c h , g l y c o l y t i c ( F G ) , a n d I = t y p e 2 B / f a s t - t w i t c h , o x i d a t i v e - g l y c o l y t i c ( F O G ) . A n a l y s i s o f the d a t a g e n e r a t e d f r o m these f i b e r c o u n t s i n c l u d e d p a r a m e t r i c ana l ys i s o f v a r i a n c e ( A N O V A ) a n d T u k e y ' s p r o c e d u r e f o r i n t e r c o m p a r i s o n s o f n o r m a l a n d d y s t r o p h i c g r o u p m e a n s . T h e s ta t i s t i ca l c o m p u t e r 76 p r o g r a m s i n the "Stats P l u s " a n d A N O V A p a c k a g e s s u p p l i e d b y H u m a n S y s t e m D y n a m i c s w e r e u s e d f o r c o m p u t a t i o n s a c c o r d i n g to s t a n d a r d s ta t i s t i ca l p r o c e d u r e s ( K l u g h , 1974) . D e t e r m i n a t i o n o f P a r v a l b u m i n C o n t e n t i ) P r o t e i n D e t e r m i n a t i o n . T o t a l s o l u b l e p r o t e i n ( T S P ) c o n c e n t r a t i o n s w e r e d e t e r m i n e d i n m u s c l e ex t rac ts c o r r e s p o n d i n g to those used f o r the R I A b y the B i o - R a d m e t h o d u s i n g p o r c i n e g a m m a g l o b u l i n as a s t a n d a r d . B o t h the 3 2 - w e e k a n d 2 - w e e k n o r m a l s a m p l e s w e r e r u n o n the s t a n d a r d ( 2 0 - 1 4 0 pg ) B i o - R a d assay , w h i l e the 2 - w e e k d y s t r o p h i c s a m p l e s r e q u i r e d the B i o - R a d m i c r o a s s a y ( 1 - 2 0 pg ) . A l l s tanda rds a n d samp les w e r e m a d e u p i n T r i s sa l i ne b u f f e r at p H 7.4 (100 m M N a C l , 20 m M T r i s base , 1 m M E D T A , 1 m M D T T , 0 . 0 2 % s o d i u m a z i d e ) . T h e assay tubes w e r e s h a k e n o n a v o r t e x , w i t h o u t f r o t h i n g , a n d w e r e a l l o w e d to s i t at r o o m t e m p e r a t u r e f o r 30 m i n . T h e p r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d , at o p t i c a l d e n s i t y at 595 ( O . D . 5 9 5 ) , o n a P h i l i p s P V 8600 s p e c t r o p h o t o m e t e r a n d the T S P c o n c e n t r a t i o n was r e a d f r o m the s t a n d a r d c u r v e ( F i g s . 21 a n d 22) . i i ) R a d i o i m m u n o a s s a y . T h e p a r v a l b u m i n c o n t e n t i n 32 a n d 2 w e e k E D L a n d so leus m u s c l e s was m e a s u r e d b y r a d i o i m m u n o a s s a y . R a b b i t a n t i - m o u s e p a r v a l b u m i n a n t i s e r u m was d e v e l o p e d i n o u r l a b o r a t o r y . T h e p a r v a l b u m i n u s e d as a s t a n d a r d i n the assay was f r o m t w o s o u r c e s . P u r i f i e d rat s ke le ta l m u s c l e p a r v a l b u m i n was p u r c h a s e d f r o m C a l b i o c h e m a n d u s e d at a n i n i t i a l d i l u t i o n o f 80 n g P V / 1 0 0 p i b u f f e r . I n a d d i t i o n , a n i n i t i a l d i l u t i o n o f 32 p l / m l o f a w h o l e m u s c l e h o m o g e n a t e f r o m m o u s e f a s t - t w i t c h m u s c l e was a lso u s e d as a s t a n d a r d . T h i s d i l u t i o n r e p r e s e n t e d a P V c o n c e n t r a t i o n o f 160 n g / 100 p i b u f f e r . I n a p r e l i m i n a r y assay , the s t a n d a r d c u r v e p r o d u c e d u s i n g th is i n i t i a l d i l u t i o n was f o u n d to c o m p a r e f a v o u r a b l y to that o f the p u r i f i e d rat p a r v a l b u m i n . T h e m u s c l e s a m p l e s that w e r e o r i g i n a l l y h o m o g e n i z e d i n a 20 t i m e s v o l u m e o f T r i s b u f f e r w e r e f u r t h e r d i l u t e d to 50 t imes v o l u m e i n the T r i s sa l i ne b u f f e r used f o r the 77 F i g u r e 21 S t a n d a r d c u r v e f o r the B i o - R a d P r o t e i n assay ( 2 0 - 1 4 0 p g ) , O . D . 5 9 5 . T o t a l s o l u b l e p r o t e i n va l ues f o r a l l the 3 2 - w e e k m u s c l e samp les w e r e e x t r a p o l a t e d f r o m th is c u r v e . 78 4 0 60 80 100 120 140 Protein Concentrat ion ug/100 ul 7<? F i g u r e 22 S t a n d a r d c u r v e f o r the B i o - R a d P r o t e i n m i c r o a s s a y ( 1 - 2 0 ug ) u s e d to c a l c u l a t e the to ta l s o l u b l e p r o t e i n i n n o r m a l a n d d y s t r o p h i c 2 - w e e k m u s c l e s a m p l e s . 80 3 - 6 9 12 15 18 21 P R O T E I N C O N C E N T R A T I O N u g / 1 0 0 u l 81 p r o t e i n assay. T h e samp les w e r e f u r t h e r d i l u t e d i n R I A b u f f e r a n d tes ted i n the f o l l o w i n g c o n c e n t r a t i o n s : N o r m a l 32 w e e k E D L 1:100, 1:200 D y s t r o p h i c 32 w e e k E D L 1:25, 1:50 N o r m a l 32 w e e k so leus 1:5, 1:10 D y s t r o p h i c 32 w e e k so leus 1:10, 1:20 A l l 2 w e e k samp les 1:5 - 1:160 A l l assays w e r e r u n i n t r i p l i c a t e as d e s c r i b e d u n d e r the g e n e r a l m e t h o d s s e c t i o n f o r the R I A . P a r v a l b u m i n c o n t e n t i n the m u s c l e s a m p l e was c a l c u l a t e d b y e x t r a p o l a t i n g a n g / 1 0 0 p i v a l u e f o r p a r v a l b u m i n at a g i v e n p e r c e n t b i n d i n g f r o m the s t a n d a r d c u r v e , a n d m u l t i p l y i n g that b y the a p p r o p r i a t e d i l u t i o n f a c t o r . 82 T A B L E I V . S u m m a r y o f f i x a t i o n a n d s e c t i o n i n g m e t h o d s i n v e s t i g a t e d f o r i m m u n o h i s t o c h e m i c a l l o c a l i z a t i o n o f p a r v a l b u m i n . F i x a t i o n E m b e d d i n g / S e c t i o n i n g M o r p h o l o g y Status P e r m e a b i l i t y to A n t i b o d v S t a i n i n g 1 0 % C a + + - F o r m a l i n aceta te - p e r f u s i o n f i x e d - p o s t - f i x e d c r y o s t a t , - f l o a t i n g a n d a d h e r e d to s l i des - p a r a f f i n - a - a + + + f r e s h t i ssue - ace tone o r e t h a n o l p o s t - f i x e d c ryos ta t — + 5 % a c r o l e i n i n P B S b u f f e r c r yos ta t p a r a f f i n E p o n - a +b ++ + + + + + 2 . 5 % g l u t a r a l d e h y d e i n 0.1 M S o r e n s e n ' s b u f f e r E p o n - b — — 0 . 5 % B e n z o q u i n o n e i n P B S b u f f e r c r yos ta t p a r a f f i n E p o n — + + — B o u i n ' s f i x a t i v e p a r a f f i n v i b r a t o m e + + +c + 4 % p a r a f o r m a l d e h y d e / 0 . 1 % g l u t a r a l d e h y d e i n 0.1 M So rensen ' s b u f f e r c r yos ta t E p o n v i b r a t o m e + + - c - f r e e z e - d r i e d / a c r o l e i n v a p o u r - f i x e d p a r a f f i n - - - f r e e z e - s u b s t i t u t i o n 1% p i c r i c a c i d i n e t h a n o l at - 7 0 ° C p a r a f f i n +d ++ ++ - p o o r , + m e d i o c r e , ++ g o o d a s h r i n k a g e o f the t issue u p o n f i n a l d e h y d r a t i o n b w r i n k l e s i n the E p o n d u e to e t c h i n g p r o c e d u r e c p r o b l e m s m o u n t i n g a n d s t a b i l i z i n g s m a l l t i ssue b l o c k s : sec t i ons too t h i c k d s o m e t i ssue a r t i f a c t c a u s e d b y e i t he r too s l o w f r e e z i n g o r a l l o w i n g t issue to d r y ou t b e f o r e e m b e d d i n g 83 R E S U L T S C h a r a c t e r i z a t i o n o f P a r v a l b u m i n T h e e l e c t r o p h o r e t i c p r o p e r t i e s o f m o u s e ske le ta l m u s c l e p a r v a l b u m i n h a v e been s h o w n to be s i m i l a r to those o f rat s ke le ta l m u s c l e p a r v a l b u m i n ( H e i z m a n n , et a l . , 1982; H e i z m a n n , 1984) . T h e c o m i g r a t i o n e x p e r i m e n t s o f p u r e ra t s k e l e t a l m u s c l e p a r v a l b u m i n w i t h n o r m a l E D L m u s c l e s a m p l e s , r e s o l v e d o n I E F gels ( F i g . 2 3 ) , s h o w e d that ra t P V c o m i g r a t e s w i t h the p r o t e i n b a n d i d e n t i f i e d as P V i n a p r e v i o u s repo r t ( J a s c h a n d M o a s e , 1985) . In a d d i t i o n , rat P V c o m i g r a t e d w i t h the b a n d i n the d y s t r o p h i c so leus s a m p l e s that was a lso b e l i e v e d to be P V ( F i g . 2 4 A ) . T h i s b a n d was no t e v i d e n t i n m u s c l e s a m p l e s f r o m the n o r m a l a d u l t so leus ( F i g . 2 4 C ) . C o m p a r i s o n o f n o r m a l a n d d y s t r o p h i c w h o l e m u s c l e samp les b y I E F ( F i g . 25) s h o w e d a d e f i n i t e dec rease i n the d e n s i t y o f the P V b a n d i n the d y s t r o p h i c E D L at 3 2 - w e e k s o f age . A d r a m a t i c i nc rease a b o v e n o r m a l l eve ls was n o t e d i n th is b a n d i n the a d u l t d y s t r o p h i c so leus at the same age. A n u m b e r o f o t h e r m u s c l e p r o t e i n s w e r e a l t e red i n the d y s t r o p h i c m u s c l e s b u t these h a v e b e e n i d e n t i f i e d a n d d i s c u s s e d i n d e t a i l i n a p r e v i o u s s t u d y ( Jasch a n d M o a s e , 1985) . B y c o m p a r i s o n , s i m i l a r I E F e x p e r i m e n t s o f the 2 - w e e k n o r m a l a n d d y s t r o p h i c m u s c l e samp les ( F i g . 26) s h o w e d no d i f f e r e n c e i n the p r o t e i n d i s t r i b u t i o n b e t w e e n n o r m a l a n d d y s t r o p h i c a n i m a l s at th is s tage. I n p a r t i c u l a r , the P V b a n d was p r o m i n e n t i n b o t h E D L s a m p l e s regard less o f g e n o t y p e , b u t i t was u n d e t e c t a b l e i n e i t he r the n o r m a l o r d y s t r o p h i c so leus m u s c l e s a m p l e s . T h e p o s i t i o n s o f the m o u s e ske le ta l m u s c l e p r o t e i n s h a v e b e e n c h a r a c t e r i z e d o n t w o - d i m e n s i o n a l ge ls b y o the r i nves t i ga to r s ( C r o w a n d K u s h m e r i c k , 1982; J a s c h a n d M o a s e , 1985) . C o m p a r i s o n o f o u r gels to those p u b l i s h e d was the bas is f o r i d e n t i f y i n g the p r o t e i n s l a b e l l e d i n F i g u r e s 27 - 30 o f the 2 - w e e k s a m p l e s . E x a m i n a t i o n o f the 2 - w e e k s a m p l e s c o n f i r m e d that P V m ig ra tes as a s i ng l e b a n d to the 14 K D m o l e c u l a r w e i g h t r ange . T h i s b a n d was c o n f i r m e d to be P V b y R I A as d e s c r i b e d i n the m e t h o d s . 84 F i g u r e 23 I soe lec t r i c f o c u s i n g ( I E F ) t ube gels c o m p a r i n g n o r m a l m o u s e E D L m u s c l e p r o t e i n (1) to p u r e rat m u s c l e p a r v a l b u m i n (2). T h e p r o t e i n b a n d ( P V ) i n the m o u s e m i g r a t e s to the same i s o e l e c t r i c p o i n t as p u r e rat p a r v a l b u m i n . C o m p l e t e c o m i g r a t i o n s tud ies o f these s a m p l e s h a v e b e e n r e p o r t e d e a r l i e r b y J a s c h a n d M o a s e (1985) . I E F gels a re o r i e n t e d w i t h the bas i c e n d to the l e f t a n d the a c i d i c e n d to the r i g h t . M o u s e m u s c l e samp les are f r o m n o r m a l 3 2 - w e e k - o l d a n i m a l s . 85 1. mouse \ — PV HUH 1 '> 2. rat PV i • » 3 8b Figure 24 Isoelectric focusing tube-gels comparing adult dystrophic (DY) and normal (N) soleus muscle proteins. Authentic rat muscle parvalbumin (PV) comigrates (A) with a protein band that appears in the dystrophic sample (B) but not in the normal sample (C), as indicated by the arrow. All gels contain proteins from 32 week mice. Acidic end to the left, basic end to the right. 87 98 F i g u r e 25 P r o t e i n b a n d s o f n o r m a l ( N ) a n d d y s t r o p h i c ( D Y ) a d u l t E D L a n d so leus ( S O L ) m u s c l e s sepa ra ted a c c o r d i n g to t h e i r i s o e l e c t r i c p o i n t o n i s o e l e c t r i c f o c u s i n g t u b e - g e l s . T h e p a r v a l b u m i n ( P V ) b a n d is i d e n t i f i e d b y an a r r o w . N o t e the a p p e a r a n c e o f th is b a n d i n the d y s t r o p h i c so leus a n d the d e c r e a s e d d e n s i t y o f the P V b a n d i n the d y s t r o p h i c E D L . I E F gels are o r i e n t e d w i t h the a c i d i c e n d to the le f t a n d the b a s i c e n d to the r i g h t . 89 © F i g u r e 26 W h o l e m u s c l e p r o t e i n d i s t r i b u t i o n , i so la ted b y i s o e l e c t r i c f o c u s i n g tube g e l e l e c t r o p h o r e s i s , f o r n o r m a l ( N ) a n d d y s t r o p h i c ( D Y ) 2 - w e e k E D L a n d so leus ( S O L ) s a m p l e s . T h e p o s i t i o n o f the p a r v a l b u m i n b a n d ( P V ) is i n d i c a t e d b y the a r r o w f o r e a c h g e l . A t 2 w e e k s o f age o n l y the E D L samp les c o n t a i n de tec tab le quan t i t i e s o f p a r v a l b u m i n . T h e d e n s i t y o f the b a n d appea rs to be s i m i l a r f o r n o r m a l a n d d y s t r o p h i c s a m p l e s . 91 91 T h e P V spo t was p r o m i n e n t i n the gels o f n o r m a l a n d d y s t r o p h i c E D L samp les ( F i g s . 27 a n d 28) b u t b a r e l y de tec tab le i n e i t he r o f the so leus ex t rac t s ( F i g s . 29 a n d 30) . T h e d i s t r i b u t i o n o f p r o t e i n s at 2 w e e k s o f age was s i m i l a r to tha t r e p o r t e d f o r the a d u l t m u s c l e s ( Jasch a n d M o a s e , 1985) . T h i s w o u l d i n d i c a t e that the a d u l t p r o f i l e has b e e n a t t a i n e d b y 2 w e e k s o f age; h o w e v e r , the a d u l t p r o t e i n c o n c e n t r a t i o n s are p r o b a b l y a c h i e v e d la te r d u r i n g pos tna ta l d e v e l o p m e n t . C h a r a c t e r i z a t i o n o f the A n t i b o d y T h e t i t re o f the a n t i s e r a aga ins t m o u s e P V was tested b y E L I S A p r o c e d u r e s , as d e s c r i b e d i n the m e t h o d s , a n d the m o n o s p e c i f i c i t y b y i m m u n o b l o t t i n g a n d R I A . T h e s o l u b l e m u s c l e p r o t e i n s w e r e i n i t i a l l y r e s o l v e d b y o n e - d i m e n s i o n a l S D S e l e c t r o p h o r e s i s ( F i g . 31a) a n d t h e n t h e y w e r e t r a n s f e r r e d to n i t r o c e l l u l o s e p a p e r f o r i m m u n o b l o t t i n g . A s c a n be seen i n F i g u r e 3 1 b , a l l o f the l o w m o l e c u l a r - w e i g h t p r o t e i n s t r a n s f e r r e d f r o m the g e l to the n i t r o c e l l u l o s e . T h e l e f t - h a n d l ane i n F i g u r e 31a c o n t a i n e d m o l e c u l a r w e i g h t m a r k e r s ; h o w e v e r , the c o r r e s p o n d i n g l ane i n the h a l f o f the ge l that was t r a n s f e r r e d ( F i g . 31b ) c o n t a i n e d 25 p g o f ra t m u s c l e P V . A s i l l u s t r a t e d i n F i g u r e s 32 a n d 3 4 a , P V a n t i s e r a d i d no t reac t w i t h a n y o the r p r o t e i n s p resen t i n the m u s c l e ex t rac t . In the a d u l t s a m p l e s , the P V a n t i s e r a r eac ted mos t i n t e n s e l y to the rat m u s c l e P V c o n t r o l , the n o r m a l g a s t r o c n e m i u s w h i c h was a lso used as a c o n t r o l , a n d the n o r m a l E D L ( F i g . 32) . A s e x p e c t e d , the s t a i n i n g was less i n tense f o r the d y s t r o p h i c E D L a n d so leus s a m p l e s . E v e n t h o u g h P V was u n d e t e c t e d i n the n o r m a l so leus b y o n e - d i m e n s i o n a l e l e c t r o p h o r e s i s ( F i g s . 31a ) , the a n t i s e r a r e a c t i o n i n d i c a t e d the p r e s e n c e o f t race a m o u n t s o f the p r o t e i n i n the s l o w - t w i t c h m u s c l e s regard less o f g e n o t y p e ( F i g . 32) . T h e resu l ts o f the R I A d e s i g n e d to test the c r o s s - r e a c t i v i t y o f the a n t i P V s e r u m w i t h C a B P a n d c a l m o d u l i n , a n d thus i ts m o n o s p e c i f i c i t y , are d e p i c t e d as s t a n d a r d c u r v e s i n F i g u r e 3 3 . O n l y the P V s t a n d a r d r esu l t ed i n c o m p l e t e d i s p l a c e m e n t 93 F i g u r e s 27 a n d 28 T w o - d i m e n s i o n a l e l e c t r o p h o r e s i s pa t te rns o f to ta l m u s c l e p r o t e i n s s e p a r a t e d a c c o r d i n g to t h e i r i s o e l e c t r i c po i n t s ( I E F ) a n d m o l e c u l a r w e i g h t s ( S D S ) , f o r 2 - w e e k n o r m a l ( N ) a n d d y s t r o p h i c ( D Y ) E D L m u s c l e s a m p l e s . E a c h I E F g e l c o n t a i n e d p r o t e i n s d e r i v e d f r o m 5 m g w e t w e i g h t m u s c l e . I E F was r u n w i t h the b a s i c e n d to the l e f t a n d the a c i d i c e n d to the r i g h t . T h e p r o m i n e n t p a r v a l b u m i n b a n d ( P V ) appea rs i n b o t h n o r m a l a n d d y s t r o p h i c samp les at a p p r o x i m a t e l y 14 K D . A c t i n ( A ) , t r o p o m y o s i n ( T M ) a n d m y o s i n l i gh t c h a i n s ( L C ) a re a lso i n d i c a t e d . 94 2 WEEK N EDL © IEF. SDS A / M J T M L C l f ^ , ; L C 2 f < ~ V L C 3 f 2 WEEK DY EDL (2^ IEF * SDS TM LC1 f _ ^ L C 2 f 95- F i g u r e s 29 a n d 30 T w o - d i m e n s i o n a l pa t te rns o f to ta l m u s c l e p r o t e i n s f r o m n o r m a l ( N ) a n d d y s t r o p h i c ( D Y ) 2 - w e e k s o l e u s , sepa ra ted o n 3 x 125 m m I E F t u b e - g e l s a n d o n g r a d i e n t S D S - P A G E a c c o r d i n g to O ' F a r r e l l (1975) . I E F was f r o m le f t (bas ic ) to r i g h t ( a c i d i c ) . T h e p a r v a l b u m i n b a n d ( P V ) is b a r e l y v i s i b l e i n b o t h n o r m a l a n d d y s t r o p h i c e l e c t r o p h o r e t i c pa t te rns . 96 2 WEEK N SOLEUS IEF SDS 2 WEEK DY SOLEUS IEF SDS TM LClg^ T L C 2 s ^ L C 2 f PV © A TM LC1 S P V T L C 2 s l £ 2 f V F i g u r e s 31 A g r a d i e n t 1 - D S D S - P A G E ( F i g . 31b ) o f a d u l t m u s c l e samp les f r o m w h i c h the p r o t e i n s w e r e t r a n s f e r r e d to n i t r o c e l l u l o s e p a p e r ( F i g . 3 2 ) , as d e s c r i b e d i n M e t h o d s . T h e s lab ge l i n F i g . 3 1 a rep resen ts the c o r r e s p o n d i n g h a l f o f the g e l that was s t a i n e d w i t h C o o m a i s s e B l u e R 2 5 0 . T h e a r r o w i n d i c a t e s the p a r v a l b u m i n b a n d a n d the lanes are i d e n t i f i e d as f o l l o w s : a) n o r m a l g a s t r o c n e m i u s ; b ) d y s t r o p h i c E D L ; c ) n o r m a l E D L ; d ) d y s t r o p h i c so leus ; e) n o r m a l so leus . F i g u r e 32 I m m u n o b l o t ( F i g . 32) o f n o r m a l a n d d y s t r o p h i c a d u l t m u s c l e h o m o g e n a t e s a m p l e s , t r a n s f e r r e d f r o m the S D S s lab gels ( F i g . 31b ) T h e i m m u n o b l o t is r e v e r s e d to the p a t t e r n s h o w n i n F i g . 3 1 a , w i t h one e x c e p t i o n . T h e e x t r e m e l e f t - h a n d lane i n F i g u r e 31a c o n t a i n s m o l e c u l a r w e i g h t m a r k e r s w h i l e the c o r r e s p o n d i n g l ane i n F i g u r e 32 c o n t a i n s p u r e rat p a r v a l b u m i n . O n l y the n o r m a l ( N ) E D L , g a s t r o c n e m i u s ( G A S T ) , a n d p a r v a l b u m i n c o n t r o l ( P V ) a re h e a v i l y s t a i n e d b y the a n t i - m o u s e p a r v a l b u m i n an t i se ra . 98 99 o f 1 2 5 I - P V b i n d i n g to the a n t i s e r u m . N o c r o s s - r e a c t i v i t y w i t h C a B P o r c a l m o d u l i n was o b s e r v e d . T h e d i s t r i b u t i o n o f s o m e o f the h i g h e r m o l e c u l a r w e i g h t s o l u b l e - p r o t e i n s was a lso a l t e red i n the d y s t r o p h i c m u s c l e at 32 w e e k s o f age. F i g u r e 34 s h o w s the b a n d i n g p a t t e r n f o r the a d u l t m u s c l e s a m p l e s . U p o n c lose e x a m i n a t i o n o f th is g e l , i t is e v i d e n t that the d i s t r i b u t i o n o f p ro te i ns ( a r r o w h e a d s ) i n the d y s t r o p h i c E D L is s i m i l a r to that o f the n o r m a l so leus . A l t h o u g h less o b v i o u s , the p a t t e r n i n the d y s t r o p h i c so leus r esemb les that o f the f a s t - t w i t c h m u s c l e s w i t h respec t to p a r t i c u l a r p r o t e i n b a n d s . O n l y P V was i d e n t i f i e d o n the ge ls . T h e o t h e r p r o m i n e n t b a n d s w e r e not c h a r a c t e r i z e d i n the p resen t s t u d y , b u t p r o b a b l y rep resen t the v a r i o u s m y o s i n l i g h t - c h a i n s , t r o p o m y o s i n , t r o p o n i n a n d a c t i n . W h e r e a s d e f i n i t e changes i n P V a n d some o f the o t h e r m u s c l e p r o t e i n s c o u l d be seen i n the a d u l t p r e p a r a t i o n s , the S D S gels a n d c o r r e s p o n d i n g i m m u n o b l o t s o f the 2 - w e e k ex t rac t s r e v e a l e d i d e n t i c a l f ea tu res w h e n c o m p a r i n g n o r m a l a n d d y s t r o p h i c s a m p l e s . A l t h o u g h d i f f e r e n c e s c o u l d be seen b e t w e e n fas t a n d s l o w - t w i t c h m u s c l e s , no d i f f e r e n c e s r e s u l t e d d u e to g e n o t y p e . T h e P V b a n d was c l e a r l y i d e n t i f i a b l e i n b o t h E D L s a m p l e s ( F i g . 3 5 b ) , a n d reac ted p o s i t i v e l y to the a n t i s e r a a f t e r i m m u n o b l o t t i n g ( F i g . 35a) . P a r v a l b u m i n was u n d e t e c t a b l e i n e i t h e r o f the so leus s a m p l e s b y S D S - P A G E ( F i g . 3 5 b ) , b u t was f a i n t l y e v i d e n t a f t e r i m m u n o b l o t t i n g ( F i g . 35a) . I m m u n o h i s t o c h e m i s t r v T h e d i s t r i b u t i o n o f P V i n m u s c l e f i b e r s was i n v e s t i g a t e d b y P A P m e t h o d s u s i n g the a n t i s e r u m aga ins t m o u s e ske le ta l m u s c l e P V , o n p a r a f f i n sec t i ons o f m u s c l e t issue f i x e d b y f r e e z e - s u b s t i t u t i o n . P a r v a l b u m i n i m m u n o r e a c t i v i t y was c a t e g o r i z e d i n t o th ree g r o u p s o f s t a i n i n g i n tens i t y : l i g h t ( L ) , d a r k ( D ) , a n d i n t e r m e d i a t e (I). T h e d a r k f i b e r s w e r e c o n s i d e r e d P V p o s i t i v e , w h e r e a s the l i g h t f i b e r s w e r e a s s u m e d to h a v e no i m m u n o r e a c t i v i t y to P V . A s u m m a r y o f the m e a n p e r c e n t a g e o f f i b e r s i n e a c h c a t e g o r y is g i v e n i n T a b l e V . M u s c l e s w e r e a lso f i b e r - t y p e d f o r m y o f i b r i l l a r A T P a s e 100 Figure 33 Standard curve for the radioimmunoassay of rat skeletal muscle parvalbumin (--o—o--), bovine cerebellum CaBP (—•--•--), and bovine brain calmodulin, CaM, (—x—x—). No cross-reactivity between parvalbumin, CaBP or calmodulin was observed. A separate abscissa has been drawn for the two brain calcium-binding proteins. 101 IOZ F i g u r e 34 G r a d i e n t ( 5 - 2 2 % ) o n e - d i m e n s i o n a l S D S - P A G E o f 3 2 - w e e k n o r m a l ( N ) a n d d y s t r o p h i c ( D Y ) m u s c l e p r o t e i n s f o r g a s t r o c n e m i u s ( G A S T ) , E D L a n d so leus . T h e p a r v a l b u m i n b a n d ( P V ) , a p p r o x i m a t e l y 14 K D , is i n d i c a t e d b y the a r r o w . T h i s g e l c o r r e s p o n d s to the i m m u n o b l o t i n F i g u r e 3 1 . D i f f e r e n c e s i n the p r o t e i n d i s t r i b u t i o n b e t w e e n the n o r m a l a n d d y s t r o p h i c , a n d f a s t - t w i t c h vs s l o w - t w i t c h m u s c l e s c a n a lso be seen i n s o m e o f the h i g h e r m o l e c u l a r w e i g h t p r o t e i n b a n d s ( a r r o w h e a d s ) . N o n e o f w h i c h b a n d s reac ted w i t h the p a r v a l b u m i n a n t i s e r a o n the i m m u n o b l o t . M o l e c u l a r w e i g h t m a r k e r s a re i n d i c a t e d i n k i l o d a l t o n s ( K D ) i n the e x t r e m e le f t c o l u m n . 103 /Of F i g u r e 35 I m m u n o l o g i c a l a n a l y s i s o f 2 - w e e k n o r m a l ( N ) a n d d y s t r o p h i c ( D Y ) w h o l e m u s c l e h o m o g e n a t e s a f t e r s e p a r a t i o n o n S D S - P A G E ( F i g . 35b ) a n d e l e c t r o p h o r e t i c t r a n s f e r to n i t r o c e l l u l o s e p a p e r ( F i g . 35a) . A u t h e n t i c rat p a r v a l b u m i n was r u n o n the h a l f o f the g e l to be b l o t t e d , as a c o n t r o l f o r the an t i se ra . T h e b a n d i n g p a t t e r n s h o w n o n the n i t r o c e l l u l o s e m e m b r a n e ( F i g . 35a) is r e v e r s e d to that seen i n the s lab g e l . N i t r o c e l l u l o s e was i n c u b a t e d w i t h a n t i - m o u s e p a r v a l b u m i n a n t i s e r u m . T h e p a r v a l b u m i n b a n d is i n d i c a t e d b y the a r r o w . B o t h n o r m a l a n d d y s t r o p h i c s a m p l e s s h o w s i m i l a r p r o t e i n d i s t r i b u t i o n s at 2 w e e k s o f age . M o l e c u l a r w e i g h t m a r k e r s a re i n d i c a t e d i n k i l o d a l t o n s ( K D ) , i n the f a r r i g h t c o l u m n o f ( F i g . 3 5 b ) . 105 lOlo a n d d i v i d e d i n t o L , D , a n d I s t a i n i n g g r o u p s . T h e s e resu l ts are g i v e n i n T a b l e V I . T h e o r i g i n a l i n t e n t i n u s i n g c o r r e s p o n d i n g ha l ves o f e a c h m u s c l e was to s i m u l t a n e o u s l y t y p e the same f i b e r s f o r P V i m m u n o r e a c t i v i t y a n d m y o s i n A T P a s e . U n f o r t u n a t e l y d u e to p r o b l e m s w i t h t issue s h r i n k a g e , f r e e z i n g a r t i f a c t s , a n d loss o f s e c t i o n s , f i b e r t y p i n g f r o m s e r i a l sec t i ons o f c o r r e s p o n d i n g ha l ves o f e a c h m u s c l e was no t p o s s i b l e . H o w e v e r , the f i b e r t y p i n g was p e r f o r m e d o n sec t i ons f r o m the same m u s c l e f o r e a c h s t a i n i n g p r o c e d u r e a n d e x p r e s s e d as the m e a n p e r c e n t a g e o f f i b e r s i n e a c h g r o u p . i ) A d u l t E D L . Sec t i ons f r o m the n o r m a l E D L m u s c l e s c o n t a i n e d a he te rogeneous m o s a i c p a t t e r n o f P V i m m u n o r e a c t i v i t y ( F i g . 36) . T h r e e m a j o r e x t r a f u s a l f i b e r t ypes c o u l d be i d e n t i f i e d that v a r i e d a c c o r d i n g to P V i m m u n o r e a c t i v i t y a n d c r o s s - s e c t i o n a l d i a m e t e r s . T h e la rges t d i a m e t e r a n d da rkes t f i b e r s c o n s t i t u t e d the h i ghes t pe rcen tage o f e x t r a f u s a l f i b e r t ypes (48 .92%) . T h e r e was a lso a p o p u l a t i o n o f s m a l l f i b e r s (15 .17%) tha t w e r e n o n - r e a c t i v e to the an t i se ra . T h e r e m a i n i n g e x t r a f u s a l f i b e r s (35 .78%) w e r e i n t e r m e d i a t e i n b o t h s t a i n i n g i n t e n s i t y a n d c r o s s - s e c t i o n a l d i a m e t e r . A g r a d i e n t o f s t a i n i n g i n tens i t i es c o u l d be o b s e r v e d i n these i n t e r m e d i a t e f i b e r s ; h o w e v e r , th is g r o u p was no t f u r t h e r s u b d i v i d e d . T h i s p a t t e r n o f s t a i n i n g was i n a g r e e m e n t w i t h that r e p o r t e d i n the rat ( C e l i o a n d H e i z m a n n , 1982) a n d I C R Z m o u s e ( H e i z m a n n et a l . , 1982) . C e l i o a n d H e i z m a n n (1982) c a t e g o r i z e d the P V - p o s i t i v e f i b e r s as t y p e 2 - f a s t f i b e r s , the l i g h t f i b e r s o f the t y p e 1 - s l o w v a r i e t y , a n d the i n t e r m e d i a t e f i b e r s as t y p e 2 - f a s t f i b e r s w i t h f a t i g u e - r e s i s t a n t c h a r a c t e r i s t i c s . W h e n th i s i m m u n o r e a c t i v i t y p r o f i l e was c o m p a r e d to the d y s t r o p h i c E D L ( F i g . 37) b o t h the n u m b e r o f d a r k l y - s t a i n e d f i b e r s (31 .42%) a n d the i n t e n s i t y o f s t a i n i n g h a d d e c r e a s e d . T h e p r o p o r t i o n o f n o n - r e a c t i v e o r l i g h t f i b e r s (24 .17%) was i n c r e a s e d as was the p e r c e n t a g e o f f i b e r s i n the i n t e r m e d i a t e g r o u p (44 .55%) . T h e d y s t r o p h i c E D L t h e r e f o r e , s h o w e d a n o v e r a l l dec rease i n P V i m m u n o r e a c t i v i t y . I n a d d i t i o n , there was a g rea te r v a r i a b i l i t y i n f i b e r s ize i n the sec t i ons f r o m d y s t r o p h i c m u s c l e . P a r v a l b u m i n i m m u n o r e a c t i v i t y was not s p e c i f i c to f i b e r s i ze as i n the n o r m a l E D L . A s 107 F i g u r e s 36 a n d 37 I m m u n o h i s t o c h e m i c a l d e m o n s t r a t i o n o f p a r v a l b u m i n i n n o r m a l ( F i g . 36) a n d d y s t r o p h i c ( F i g . 3 7 ) E D L m u s c l e s f r o m 3 2 - w e e k - o l d m i c e . M u s c l e s w e r e f i x e d b y f r e e z e - s u b s t i t u t i o n , e m b e d d e d i n p a r a f f i n , a n d p r o c e s s e d as d e s c r i b e d i n M e t h o d s . T h r e e p r o m i n e n t s t a i n i n g i n tens i t i es c a n be i d e n t i f i e d : l a rge d i a m e t e r , d a r k f i b e r s ( D ) , f i b e r s o f i n t e r m e d i a t e s i ze a n d s t a i n i n g (I), a n d s m a l l n o n - r e a c t i v e o r l i g h t f i b e r s ( L ) . T h e r e are a g rea te r n u m b e r o f l i g h t f i b e r s i n the d y s t r o p h i c E D L , a n d a n a l t e r a t i o n i n f i b e r d i a m e t e r w h e n c o m p a r e d to i ts n o r m a l c o u n t e r p a r t . P e r i p h e r a l n e r v e f a s c i c l e s d i d not s t a i n . x 4 0 0 108 (09 c a n be seen i n F i g u r e 3 7 , s m a l l - d i a m e t e r f i b e r s m a y be e i t h e r L , D , o r I i n s t a i n i n g f o r P V . T h e T u k e y ' s s ta t i s t i ca l ana l ys i s p r o c e d u r e f o r i n t e r c o m p a r i s o n s i n d i c a t e d a s i g n i f i c a n t i n t e r a c t i o n e f f e c t b e t w e e n g e n o t y p e a n d P V s t a i n i n g p a t t e r n f o r the E D L sec t i ons . T h e s e f i b e r g r o u p i n g s c o r r e s p o n d e d to the m y o f i b r i l l a r A T P a s e s t a i n i n g p r o f i l e ( F i g s . 38 a n d 39) i n w h i c h the la rgest a n d pa les t f i b e r s w e r e t y p e 2 - f a s t f i b e r s , the i n t e r m e d i a t e f i b e r s w e r e t y p e 2 f i b e r s w i t h b o t h fas t a n d s l o w c h a r a c t e r i s t i c s , a n d the sma l l es t a n d d a r k e s t f i b e r s w e r e o f the s l o w v a r i e t y . T h e m y o f i b r i l l a r A T P a s e s t a i n i n g p a t t e r n was a lmos t a reve rse to that seen w i t h the P V i m m u n o h i s t o c h e m i s t r y ( T a b l e V I I ) . T h e la rge fas t t y p e f i b e r s w e r e d a r k a f t e r P V a n t i s e r a t r ea tmen t a n d l i gh t a f t e r m y o s i n A T P a s e . T h e s m a l l t ype 1 s l o w f i b e r s w e r e n o n - r e a c t i v e f o r P V b u t w e r e d a r k l y s t a i n e d b y the m y o s i n A T P a s e p r o c e d u r e . In th is s t u d y there was not a c o m p l e t e c o r r e l a t i o n b e t w e e n P V i m m u n o r e a c t i v i t y a n d m y o s i n A T P a s e s t a i n i n g . A l l t y p e 2 fas t f i b e r s c o n t a i n e d P V w h e r e a s there was a g r a d a t i o n o f P V i n tens i t i es i n the s e c o n d c lass o f t ype 2 f i b e r s r a n g i n g f r o m n o n - r e a c t i v e to i n tense . N o n e o f the s m a l l t ype 1 s l o w f i b e r s c o n t a i n e d P V . It was e v i d e n t w h e n c o m p a r i n g the pe rcen tages o b t a i n e d f r o m b o t h the P A P a n d m y o s i n A T P a s e s t a i n i n g p r o c e d u r e s ( T a b l e s V a n d V I , r e s p e c t i v e l y ) that the e x t r a f u s a l f i b e r s c o u l d no t be c a t e g o r i z e d i n t o th ree d i s t i n c t f i b e r t ypes b a s e d o n t he i r P V i m m u n o r e a c t i v i t y . W h e n t y p i n g the f i b e r s a c c o r d i n g to P V l o c a l i z a t i o n , a g rea te r pe rcen tage o f f i b e r s f e l l i n t o the I / t ype 2 a n d L / t y p e 1 g r o u p s a n d a l o w e r p e r c e n t a g e i n t o the D / t y p e 2 g r o u p t h a n was o b s e r v e d u s i n g the c o r r e s p o n d i n g m y o s i n A T P a s e r e a c t i v i t y as a bas is f o r c o m p a r i s o n . I n o t h e r w o r d s , b a s e d o n P V d i s t r i b u t i o n , the n o r m a l a n d d y s t r o p h i c E D L a p p e a r to be s l o w e r t ypes o f m u s c l e s t han is i n d i c a t e d b y the m y o s i n A T P a s e l o c a l i z a t i o n . T h e a d u l t d y s t r o p h i c E D L ( F i g . 39) s h o w e d an i nc rease i n the n u m b e r o f I / t ype 2 f i b e r s a n d a c o n c o m i t a n t dec rease i n the n u m b e r o f L / t y p e 2 e x t r a f u s a l f i b e r s w h e n t y p e d a c c o r d i n g to m y o s i n A T P a s e . A l t h o u g h no t v i s i b l e i n F i g u r e 3 9 , there was a lso 110 F i g u r e s 38 a n d 39 H i s t o c h e m i c a l d e m o n s t r a t i o n o f m y o f i b r i l l a r A T P a s e at p H 4.6 i n f r o z e n sec t i ons o f n o r m a l ( F i g . 38) a n d d y s t r o p h i c ( F i g . 39) a d u l t E D L m u s c l e s . T h r e e s t a i n i n g i n tens i t i es a re n o t e d i n the e x t r a f u s a l f i b e r s : l i g h t ( L ) , d a r k ( D ) a n d i n t e r m e d i a t e (I). T h e o u t e r , c a p s u l e a n d B a g 2 i n t r a f u s a l f i b e r (as te r i sk ) o f a m u s c l e s p i n d l e c u t t h r o u g h the p o l a r r e g i o n ( a r r o w ) a p p e a r d a r k l y s t a i n e d i n the d y s t r o p h i c E D L . A l s o no te the c e n t r a l n u c l e a t i o n a n d v a r i a t i o n i n e x t r a f u s a l f i b e r d i a m e t e r s i n the d y s t r o p h i c m u s c l e . x 6 5 0 111  an i n c r e a s e i n the n u m b e r o f D / t y p e 1 f i b e r s . A n i n c r e a s e i n the p r o p o r t i o n o f i n t e r m e d i a t e f i b e r s was cons i s ten t w i t h the P V resu l t s . O n e o f the i n t r a f u s a l f i b e r s o f m u s c l e s p i n d l e s , p r e s u m a b l y the bag2 f i b e r , was d a r k l y s t a i n e d f o r m y o s i n A T P a s e r e a c t i v i t y as was the t h i c k ou te r c a p s u l e ( F i g . 39) . i i ) A d u l t So leus . M u s c l e sec t ions o f the n o r m a l a d u l t so leus t rea ted w i t h the P V a n t i s e r a ( F i g . 40 ) w e r e p r e d o m i n a n t l y m a d e u p o f l a rge n o n - r e a c t i v e f i b e r s (76.73%) a n d a s m a l l p o p u l a t i o n o f b o t h i n t e r m e d i a t e l y (14 .60%) a n d d a r k l y (8 .39%) s t a i n e d e x t r a f u s a l f i b e r s . I n the d y s t r o p h i c so leus ( F i g . 4 1 ) , a s i g n i f i c a n t (p <.01) dec rease i n the p e r c e n t a g e o f l i g h t f i b e r s (26 .64%) , a n d a n i n c r e a s e i n the p e r c e n t a g e o f b o t h d a r k (35 .11%) a n d i n t e r m e d i a t e (44.34%) f i b e r s w e r e o b s e r v e d w h e n c o m p a r e d to the n o r m a l so leus . T h e r e a p p e a r e d to be a g rea te r n u m b e r o f s m a l l e r - d i a m e t e r f i b e r s i n the sec t i ons f r o m the d y s t r o p h i c m u s c l e , a n d these s m a l l f i b e r s w e r e u s u a l l y P V - p o s i t i v e . O v e r a l l , the re was a g rea te r p r o p o r t i o n o f f i b e r s p o s i t i v e f o r P V i n the d y s t r o p h i c so leus t h a n i n the n o r m a l so leus . T h e mos t s t r i k i n g o b s e r v a t i o n was the s i m i l a r i t y i n the P V l o c a l i z a t i o n i n the e x t r a f u s a l f i b e r s o f the d y s t r o p h i c so leus a n d the d y s t r o p h i c E D L . T h e m e a n p e r c e n t a g e o f f i b e r s c a t e g o r i z e d i n t o e a c h o f the s t a i n i n g g r o u p s was v i r t u a l l y i d e n t i c a l . U n l i k e t h e i r n o r m a l c o u n t e r p a r t s , the d i s t r i b u t i o n o f e x t r a f u s a l m u s c l e f i b e r s e i t h e r p o s i t i v e l y o r n e g a t i v e l y s t a i n e d f o r P V was e v e n l y d i s p e r s e d i n the sec t i ons f r o m the d y s t r o p h i c E D L a n d so leus ( T a b l e V ) . T h i s t r e n d to a m o r e h o m o g e n e o u s p o p u l a t i o n o f e x t r a f u s a l f i b e r t ypes i n the 3 2 - w e e k d y s t r o p h i c so leus was a lso o b s e r v e d i n the m y o s i n A T P a s e s t a i n i n g . It is e v i d e n t f r o m b o t h F i g u r e s 42 a n d 4 3 , as w e l l as f r o m T a b l e V I , that the d y s t r o p h i c so leus has a g rea te r p r o p o r t i o n o f i n t e r m e d i a t e f i b e r s t h a n i ts n o r m a l c o u n t e r p a r t . In a d d i t i o n to the c o n v e r s i o n i n s t a i n i n g i n tens i t i es there was a g rea te r v a r i a b i l i t y i n the e x t r a f u s a l f i b e r d i a m e t e r s i n the s e c t i o n o f the d y s t r o p h i c so leus t h a n i n the s e c t i o n o f the n o r m a l t i ssue. A c o r r e l a t i o n b e t w e e n f i b e r s i ze a n d f i b e r t y p e c o u l d no t be 113 F i g u r e s 40 a n d 41 P a r a f f i n sec t i ons o f f r e e z e - s u b s t i t u t e d m u s c l e s f r o m n o r m a l ( F i g . 40 ) a n d d y s t r o p h i c ( F i g . 41 ) a d u l t so leus m u s c l e s . Sec t i ons w e r e p r o c e s s e d f o r p a r v a l b u m i n i m m u n o h i s t o c h e m i s t r y as d e s c r i b e d i n the M e t h o d s . T h r e e s t a i n i n g i n tens i t i es are i n d i c a t e d , l i g h t ( L ) , d a r k ( D ) a n d i n t e r m e d i a t e (I). T h e r e appea rs to be a n i n c r e a s e d n u m b e r o f i n t e r m e d i a t e f i b e r s , a n d a g rea te r v a r i a t i o n i n f i b e r s i ze i n the d y s t r o p h i c so leus w h e n c o m p a r e d to i ts n o r m a l c o u n t e r p a r t . x 6 5 0 114 IIS F i g u r e s 42 a n d 43 F r o z e n sec t i ons o f n o r m a l ( F i g . 42) a n d d y s t r o p h i c ( F i g . 43 ) a d u l t so leus m u s c l e s , s t a i n e d f o r m y o f i b r i l l a r A T P a s e p H 4 .6 . T h e th ree s t a i n i n g i n t ens i t i e s are l a b e l l e d l i g h t ( L ) , d a r k ( D ) a n d i n t e r m e d i a t e (I). T h e d y s t r o p h i c so leus c o n t a i n s m a n y s m a l l d i a m e t e r e x t r a f u s a l f i b e r s w h i c h v a r y i n s t a i n i n g i n t e n s i t y . O v e r a l l t he re appears to be f e w e r d a r k f i b e r s a n d m o r e l i g h t f i b e r s t h a n o b s e r v e d i n the n o r m a l so leus . A p o l a r m u s c l e s p i n d l e ( a r r o w s ) is i n d i c a t e d i n b o t h sec t i ons . T h e B a g 2 i n t r a f u s a l f i b e r (as ter isks) s ta ins d a r k l y i n b o t h n o r m a l a n d d y s t r o p h i c m u s c l e s , h o w e v e r , o n l y the o u t e r c a p s u l e o f the s p i n d l e i n the d y s t r o p h i c s e c t i o n is h e a v i l y s t a i n e d . x 6 5 0 116 in m a d e s i n c e a l l th ree s t a i n i n g i n tens i t i es w e r e o b s e r v e d regard less o f the d i a m e t e r o f the m y o f i b e r . G e n e r a l l y , m u s c l e s p i n d l e s w e r e less e v i d e n t i n the sec t i ons f r o m the a d u l t m u s c l e s u s i n g P A P i m m u n o h i s t o c h e m i s t r y t h a n w i t h m y o s i n A T P a s e h i s t o c h e m i s t r y . A s i n the 3 2 - w e e k E D L , one o f the i n t r a f u s a l f i b e r s i n the sec t i ons o f the so leus musc les was d a r k l y s t a i n e d f o r m y o s i n A T P a s e a c t i v i t y ( F i g s . 42 a n d 43 ) . T h e t h i c k ou te r c a p s u l e o f m u s c l e s p i n d l e s was a lso d a r k l y s t a i n e d ( F i g . 43 ) ; h o w e v e r , th is was o n l y o b s e r v e d i n the d y s t r o p h i c t i ssue. T h e i n t r a f u s a l f i b e r s t y p i c a l l y h a d no P V i m m u n o r e a c t i v i t y i n e i t h e r n o r m a l o r d y s t r o p h i c a n i m a l s , h o w e v e r , a n o c c a s i o n a l p o s i t i v e l y - s t a i n e d i n t r a f u s a l f i b e r was o b s e r v e d i n s o m e s p i n d l e s . I m m u n o h i s t o c h e m i c a l s t a i n i n g o f s e r i a l sec t i ons t a k e n t h r o u g h o u t the l e n g t h o f a m u s c l e s p i n d l e w e r e not p e r f o r m e d . It is p o s s i b l e that r e g i o n a l d i f f e r e n c e s i n P V i m m u n o r e a c t i v i t y m a y ex i s t i n the i n t r a f u s a l f i b e r s as was seen i n C h a p t e r 1 w i t h the m y o s i n A T P a s e s t a i n i n g . i i i ) T w o w e e k E D L . T h e d i s t r i b u t i o n o f P V was s i m i l a r i n the 2 - w e e k - o l d n o r m a l E D L ( F i g . 44 ) a n d d y s t r o p h i c E D L ( F i g . 4 5 ) , a l t h o u g h the sec t i ons f r o m the d y s t r o p h i c E D L s h o w e d a s l i g h t l y g rea te r pe rcen tage o f l i g h t f i b e r s ( 2 7 % c o m p a r e d to 19%) as o u t l i n e d i n T a b l e V . T h e m a j o r i t y o f e x t r a f u s a l f i b e r s i n b o t h the n o r m a l a n d d y s t r o p h i c E D L w e r e p o s i t i v e f o r P V (50% a n d 4 1 % , r e s p e c t i v e l y ) . A s c a n be seen i n F i g u r e 4 5 , the o v e r a l l b a c k g r o u n d l e v e l o f s t a i n i n g was h i g h e r i n the d y s t r o p h i c E D L sec t i ons . T h e P V d i s t r i b u t i o n i n the neona ta l f a s t - t w i t c h m u s c l e s r e s e m b l e d that seen i n the n o r m a l a d u l t E D L . A t 2 w e e k s o f age , these m u s c l e s d i d no t s h o w the v a r i a t i o n i n f i b e r d i a m e t e r s o b s e r v e d i n the a d u l t m u s c l e s . I n t r a f u s a l f i b e r s that s t a i n e d p o s i t i v e l y f o r P V w e r e o f t e n o b s e r v e d i n p o l a r r eg ions o f m u s c l e s p i n d l e s o f the 2 - w e e k sec t i ons ( F i g s . 45 a n d 49 ) . S p i n d l e s s e c t i o n e d t h r o u g h the e q u a t o r i a l a n d j u x t a e q u a t o r i a l r e g i o n s r e v e a l e d no P V i m m u n o r e a c t i v i t y i n the i n t r a f u s a l f i b e r s ( F i g s . 44 a n d 48 ) . J u d g i n g b y the s i ze o f the i n t r a f u s a l f i b e r s that w e r e p o s i t i v e f o r P V , i t a p p e a r e d that one o f the n u c l e a r bag f i b e r s c o n t a i n e d P V ( F i g . 49 ) as w e l l as a l l o f the n u c l e a r c h a i n f i b e r s . T h i s f i n d i n g 118 is c o n t r a r y to that r e p o r t e d i n the a d u l t E D L o f the rat ( C e l i o a n d H e i z m a n n , 1982) . T h e d i s t r i b u t i o n o f e x t r a f u s a l m u s c l e f i b e r s t y p e d a c c o r d i n g to m y o s i n A T P a s e a c t i v i t y ( T a b l e V I ) was s i m i l a r i n the n o r m a l ( F i g . 46) a n d d y s t r o p h i c ( F i g . 47 ) E D L . A p p r o x i m a t e l y 8 6 % o f the e x t r a f u s a l f i b e r s w e r e l i g h t l y s t a i n e d a n d t h e r e f o r e o f the fas t ( t ype 2) v a r i e t y . F e w i n t e r m e d i a t e l y - s t a i n e d f i b e r s w e r e o b s e r v e d i n e i t he r the n o r m a l o r d y s t r o p h i c 2 - w e e k E D L . Sec t i ons f r o m the n o r m a l E D L o f t e n s ta i ned d a r k e r o v e r a l l t h a n those f r o m the d y s t r o p h i c m u s c l e s . W h e n c o m p a r e d to the a d u l t t i ssue s e c t i o n s , the m y o s i n A T P a s e s t a i n i n g p a t t e r n o f the 2 - w e e k s a m p l e s d i f f e r e d w i t h respec t to the d i s t r i b u t i o n o f i n t e r m e d i a t e a n d d a r k m u s c l e f i b e r s . T h e p r o p o r t i o n o f these t w o g r o u p s o f f i b e r s was r e v e r s e d f r o m that seen i n the a d u l t . In the sec t i ons f r o m the y o u n g e r a n i m a l s , the re was a g rea te r p e r c e n t a g e o f d a r k o r s l o w f i b e r s a n d a l o w e r p e r c e n t a g e o f i n t e r m e d i a t e f i b e r s t h a n c a l c u l a t e d f o r the a d u l t E D L . I n t r a f u s a l f i b e r s i n p o l a r r eg ions o f m u s c l e s p i n d l e s i n e i t h e r the n o r m a l ( F i g . 46 ) o r d y s t r o p h i c ( F i g . 47 ) 2 - w e e k E D L s t a i n e d a c c o r d i n g to that d e s c r i b e d i n C h a p t e r 1 f o r m y o s i n A T P a s e u n d e r a c i d p r e i n c u b a t i o n c o n d i t i o n s . T h e s m a l l d i a m e t e r i n t r a f u s a l f i b e r s , p r e s u m a b l y c h a i n f i b e r s , w e r e u n s t a i n e d w h i l e the l a r g e - d i a m e t e r i n t r a f u s a l f i b e r s w e r e d a r k l y o r i n t e r m e d i a t e l y s t a i n e d . i v ) T w o W e e k S o l e u s . T h e m a j o r i t y o f the e x t r a f u s a l m u s c l e f i b e r s i n b o t h the n o r m a l ( F i g . 48) a n d d y s t r o p h i c ( F i g . 49 ) so leus w e r e n o n - r e a c t i v e f o r P V . T h e r e was a s m a l l p o p u l a t i o n o f e x t r a f u s a l f i b e r s , h o w e v e r , that s t a i n e d p o s i t i v e l y f o r P V . T h e resu l ts f o r the so leus m u s c l e s are c o m p i l e d i n T a b l e V . N o s i g n i f i c a n t d i f f e r e n c e s w e r e n o t e d i n the s t a i n i n g pa t t e rn b e t w e e n the n o r m a l a n d d y s t r o p h i c so leus at 2 w e e k s o f age . T h e d i s t r i b u t i o n o f P V i n the e x t r a f u s a l f i b e r s was v e r y s i m i l a r to that n o t e d i n the n o r m a l a d u l t so l eus , b u t i t was d r a m a t i c a l l y d i f f e r e n t f r o m that o b s e r v e d i n the d y s t r o p h i c a d u l t so leus . P a r v a l b u m i n i m m u n o r e a c t i v i t y i n the n o r m a l m u s c l e , t h e r e f o r e , v a r i e s l i t t l e a f t e r 2 w e e k s o f age i n the so leus . In the d y s t r o p h i c so leus , 119 F i g u r e s 44 a n d 45 I m m u n o h i s t o c h e m i c a l l o c a l i z a t i o n o f p a r v a l b u m i n i n f r e e z e - s u b s t i t u t e d p a r a f f i n - sec t i ons o f n o r m a l ( F i g . 44 ) a n d d y s t r o p h i c ( F i g . 45) neona ta l E D L m u s c l e s . T h e th ree m a j o r t ypes o f f i b e r s a re l a b e l l e d l i gh t ( L ) , d a r k ( D ) , o r i n t e r m e d i a t e (I) i n s t a i n i n g . M u s c l e s p i n d l e s (a r rows ) a re i n d i c a t e d i n b o t h sec t i ons . N o t e tha t i n t r a f u s a l f i b e r s i n the d y s t r o p h i c s p i n d l e a p p e a r p o s i t i v e f o r p a r v a l b u m i n . T h r e e m o r e s p i n d l e s (not l a b e l l e d ) a re p resen t at the l o w e r edge o f F i g . 30 . T h e r e appea rs to be a r e l a t i v e l y la rge p r o p o r t i o n o f l i g h t l y s t a i n e d e x t r a f u s a l f i b e r s i n b o t h the n o r m a l a n d d y s t r o p h i c E D L at 2 w e e k s . x 6 5 0 120  F i g u r e s 46 a n d 47 F r o z e n sec t i ons o f n o r m a l ( F i g . 46 ) a n d d y s t r o p h i c ( F i g . 47 ) n e o n a t a l E D L m u s c l e s , s t a i n e d f o r m y o f i b r i l l a r A T P a s e p H 4 .6 . T h e m a j o r i t y o f the e x t r a f u s a l f i b e r s at th is age a p p e a r to be l i g h t ( L ) i n s t a i n i n g . T h e B a g 2 i n t r a f u s a l f i b e r (as te r i sks ) is d a r k l y s t a i n e d i n the m u s c l e s p i n d l e (a r rows ) o f b o t h the n o r m a l a n d d y s t r o p h i c E D L . x 6 5 0 122 (15 P V l o c a l i z a t i o n v a r i e d w i t h age , i n d i c a t i n g a n i n t e r a c t i o n b e t w e e n s t a i n i n g p a t t e r n , age a n d g e n o t y p e . I n t r a f u s a l m u s c l e f i b e r s f r o m the j u x t a e q u a t o r i a l z o n e o f m u s c l e s p i n d l e s d i d not s ta i n f o r P V ( F i g . 48 ) . P o l a r r eg ions o f m u s c l e s p i n d l e s , h o w e v e r , c o n t a i n e d i n t r a f u s a l f i b e r s w h i c h w e r e p o s i t i v e f o r P V ( F i g . 49 ) . T h e d i a m e t e r s o f these i n t r a f u s a l f i b e r s sugges ted that b o t h n u c l e a r b a g a n d n u c l e a r c h a i n f i b e r s m a y c o n t a i n P V i n the n e o n a t a l m u s c l e . I n t e r e s t i n g l y , m o r e P V was o c c a s i o n a l l y o b s e r v e d i n one o f the bag f i b e r s t h a n i n the c h a i n f i b e r s ( F i g . 49) . E x t r a f u s a l f i b e r s i n b o t h the n o r m a l ( F i g . 50) a n d d y s t r o p h i c ( F i g . 51) 2 - w e e k so leus c o u l d be c a t e g o r i z e d i n t o the th ree s t a i n i n g g r o u p s a c c o r d i n g to m y o s i n A T P a s e r e a c t i v i t y . R e g a r d l e s s o f g e n o t y p e , the p r e d o m i n a n t f i b e r c l a s s i f i c a t i o n was the L / t y p e 2 v a r i e t y ( T a b l e V I ) . T h e d y s t r o p h i c so leus c o n t a i n e d s i g n i f i c a n t l y m o r e I / t ype 2 f i b e r s t h a n i ts n o r m a l c o u n t e r p a r t b u t a s i m i l a r m e a n p e r c e n t a g e o f the D / t y p e 1 v a r i e t y . T h e p e r c e n t a g e o f D / t y p e 1 f i b e r s was l o w e r t h a n that o b s e r v e d i n the sec t i ons f r o m the n o r m a l a d u l t so leus . I n t r a f u s a l f i b e r s s t a i n e d i n a s i m i l a r f a s h i o n to that o b s e r v e d i n the E D L at 2 w e e k s o f age . S u m m a r y o f I m m u n o h i s t o c h e m i c a l R e s u l t s T h e s i g n i f i c a n t d i f f e r e n c e s i n P V a n d m y o s i n A T P a s e s t a i n i n g w e r e no t d u e to a m a i n e f f e c t o f the s ta i n b u t to a n i n t e r a c t i o n o f g e n o t y p e , age a n d m u s c l e (p <.001 a c c o r d i n g to the ana l ys i s o f v a r i a n c e ) . T h e P V d i s t r i b u t i o n p a t t e r n appea rs to v a r y a c c o r d i n g to g e n o t y p e ( m o r e t y p e I f i b e r s i n d y s t r o p h i c m u s c l e s ) , m u s c l e ( m o r e t ype D f i b e r s i n the E D L t h a n i n the so leus ) , a n d age. F o r a g i v e n m u s c l e , the l o c a l i z a t i o n o f P V a p p e a r e d to v a r y a c c o r d i n g to g e n o t y p e as w e l l as age o f the a n i m a l . T h e s e i n t e r a c t i o n e f f e c t s w e r e a lso o b s e r v e d i n the m y o s i n A T P a s e resu l t s . P a r v a l b u m i n i m m u n o h i s t o c h e m i s t r y r esu l t ed i n a g r a d a t i o n o f s t a i n i n g i n tens i t i es that m a d e i t d i f f i c u l t to t y p e the e x t r a f u s a l m u s c l e f i b e r s i n t o the 3 d i s t i n c t g r o u p s o b s e r v e d u s i n g the m y o s i n A T P a s e m e t h o d . N o t rue c o r r e l a t i o n c o u l d be d r a w n 124 F i g u r e s 48 a n d 4 9 L i g h t m i c r o g r a p h s o f p a r a f f i n sec t ions o f n o r m a l ( F i g . 48 ) a n d d y s t r o p h i c ( F i g . 49) n e o n a t a l so leus m u s c l e s , s t a i n e d f o r p a r v a l b u m i n . L i g h t ( L ) , d a r k ( D ) , o r i n t e r m e d i a t e (I) f i b e r s a re c o m m o n to b o t h n o r m a l a n d d y s t r o p h i c sec t i ons ; h o w e v e r , the b a c k g r o u n d s t a i n i n g is h i g h e r i n F i g . 49 . A m u s c l e s p i n d l e i n the j u x t a e q u a t o r i a l zone is i n d i c a t e d i n F i g . 48 ( a r r o w ) a n d a p o l a r r e g i o n s p i n d l e i n F i g . 4 9 . N o t e that one i n t r a f u s a l f i b e r is d a r k l y s t a i n e d i n the d y s t r o p h i c s p i n d l e b u t those i n the n o r m a l s p i n d l e are n o n - r e a c t i v e . A n i n t r a m u s c u l a r n e r v e i n the u p p e r r i g h t - h a n d c o r n e r o f F i g u r e 49 a lso shows s o m e i m m u n o l a b e l l i n g . x 6 5 0 125 lib Figures 50 and 51 Light micrographs of frozen sections of normal (Fig. 50) and dystrophic (Fig. 51) neonatal soleus muscles, stained for myofibrillar ATPase pH 4.6. Three types of muscle fibers are identified, light (L), dark (D), or intermediate (I), as well as a muscle spindle (arrow). There appears to be more intermediately-stained fibers in the dystrophic soleus at 2 weeks of age when compared to its normal counterpart. x650 127  T A B L E V . D i s t r i b u t i o n o f f i b e r t ypes a c c o r d i n g to p a r v a l b u m i n i m m u n o r e a c t i v i t y f o r n o r m a l ( N ) a n d d y s t r o p h i c ( D Y ) m u s c l e s at 32 a n d 2 w e e k s o f age . 32 W e e k 2 W e e k E D L S O L E U S E D L S O L E U S N D Y N D Y N D Y N D Y L i g h t 15.17 24 .17 76 .73b 26 .64 19.00 27 .07 68 .72 73 .34 D a r k 4 8 . 9 2 a 31.42 8 .39b 35.11 50 .00a 41 .00 10.50 9.46 I n t e r - 35 .78 44 .55 14.60b 44 .34 30 .75 31.98 21 .13 15.99 m e d i a t e a I nd i ca tes s ta t i s t i ca l s i g n i f i c a n c e b e t w e e n n o r m a l a n d d y s t r o p h i c va l ues at p < 0 .05 . b Ind i ca tes s ta t i s t i ca l s i g n i f i c a n c e b e t w e e n n o r m a l a n d d y s t r o p h i c va l ues at p < 0 .01 . 129 T A B L E V I . D i s t r i b u t i o n o f f i b e r t ypes a c c o r d i n g to m y o s i n A T P a s e r e a c t i v i t y f o r n o r m a l ( N ) a n d d y s t r o p h i c ( D Y ) musc les at 32 a n d 2 w e e k s o f age . 32 W e e k 2 W e e k E D L S O L E U S E D L S O L E U S N D Y N D Y N D Y N D Y L / t y p e 2 6 9 . 8 8 b 17.60 50 .25b 18.60 85.38 86 .03 68 .28 54.52 D / t y p e 1 1.50 5.10 4 2 . 3 7 b 15.09 11.36 13.28 27 .95 32 .15 5 / t y p e 2 2 8 . 6 2 b 77.27 7 .33b 66 .33 3.21 0.63 4.08 13.30 a I nd i ca tes s ta t i s t i ca l s i g n i f i c a n c e b e t w e e n n o r m a l a n d d y s t r o p h i c va lues at p < 0 .05 . b I nd i ca tes s ta t i s t i ca l s i g n i f i c a n c e b e t w e e n n o r m a l a n d d y s t r o p h i c va lues at p < 0 .01 . 130 TABLE VII. Correlation of extrafusal fiber classifications for parvalbumin and myosin ATPase staining procedures. Light Dark Intermediate Parvalbumin non-reactive high medium slow fast/type2 fast or slow i Myosin fast slow fast-oxidative ATPase t y p e 2 typel t y p e 2 131 b e t w e e n P V a n d m y o s i n A T P a s e r e a c t i v i t y . T h e d i s t r i b u t i o n o f t y p e 1 a n d t y p e 2 f i b e r s t y p e d a c c o r d i n g to m y o s i n A T P a s e d i f f e r e d f r o m the d i s t r i b u t i o n o f n o n - r e a c t i v e a n d r e a c t i v e f i b e r s d e t e r m i n e d b y the P V s t a i n i n g . W h e n the e x t r a f u s a l m u s c l e f i b e r s w e r e c a t e g o r i z e d a c c o r d i n g to P V i m m u n o r e a c t i v i t y , b o t h the E D L a n d so leus c o n t a i n e d an o v e r a l l h i g h e r p r o p o r t i o n o f s l o w f i b e r t ypes t h a n was o b s e r v e d u s i n g the m y o s i n A T P a s e s t a i n . T h i s o b s e r v a t i o n is b a s e d o n the f a c t tha t f i b e r s o f the t y p e 1 / S O v a r i e t y h a d no P V i m m u n o r e a c t i v i t y ( C e l i o a n d H e i z m a n n , 1982) . A s e x p e c t e d , the E D L c o n t a i n e d m o r e f i b e r s that w e r e p o s i t i v e f o r p a r v a l b u m i n t h a n the so leus . W h e r e a s the d i s t r i b u t i o n o f p a r v a l b u m i n d i d no t v a r y s i g n i f i c a n t l y w i t h age i n the n o r m a l m u s c l e s , a s i g n i f i c a n t d i f f e r e n c e was o b s e r v e d i n the l o c a l i z a t i o n o f P V i n the a d u l t d y s t r o p h i c m u s c l e s w h e n c o m p a r e d to t h e i r n o r m a l c o u n t e r p a r t s . T h e P V s t a i n i n g p a t t e r n was v e r y s i m i l a r b e t w e e n the a d u l t d y s t r o p h i c E D L a n d d y s t r o p h i c so leus . P a r v a l b u m i n C o n t e n t L e v e l s o f P V w e r e m e a s u r e d b y R I A u s i n g a n t i - m o u s e s k e l e t a l m u s c l e P V a n t i s e r u m r a i s e d i n the r a b b i t . I n d i v i d u a l m u s c l e s w e r e p o o l e d f r o m a n u m b e r o f a n i m a l s i n e a c h g r o u p to o b t a i n e n o u g h m u s c l e ex t r ac t i n e a c h s a m p l e f o r the i m m u n o a s s a y . T y p i c a l s t a n d a r d c u r v e s o f the assay f o r P V f o r the a d u l t a n d 2 - w e e k samp les are s h o w n i n F i g u r e s 52 a n d 5 3 , r e s p e c t i v e l y . In the ra t s y s t e m the 1 2 5 I - P V , a n t i s e r u m a n d p a r v a l b u m i n s t a n d a r d w e r e a l l d e r i v e d f r o m rat s k e l e t a l m u s c l e . W h e n d i l u t i o n s o f m o u s e m u s c l e homogena tes w e r e a d d e d to the rat s y s t e m i n c o m p l e t e c r o s s - r e a c t i v i t y o c c u r r e d , i n d i c a t i n g re l a t i ve spec ies s p e c i f i c i t y . T h e r e f o r e , i n o r d e r to d e t e r m i n e the P V c o n c e n t r a t i o n i n the m o u s e m u s c l e s a m p l e s , a n d i n v i e w o f the f a c t that o n l y p u r e ra t P V was a v a i l a b l e f o r i o d i n a t i o n , a he te rogeneous assay was u s e d . T h e i o d i n a t e d ra t P V was i n c u b a t e d w i t h a n t i - m o u s e P V a n t i s e r u m a n d a s t a n d a r d c u r v e c o n s t r u c t e d u s i n g d i l u t i o n s o f the c a l i b r a t e d m o u s e m u s c l e h o m o g e n a t e . C a l i b r a t i o n o f th is s a m p l e was d o n e b y e x t r a p o l a t i n g the m i d - p o i n t f r o m the m o u s e 132 h o m o g e n a t e d i l u t i o n c u r v e to the m i d - p o i n t o f the rat s t a n d a r d c u r v e . T h i s v a l u e was u s e d to d e t e r m i n e the r e a l c o n c e n t r a t i o n o f P V at a g i v e n d i l u t i o n . W h i l e th is p r o c e d u r e m a y i n t r o d u c e s o m e i n a c c u r a c i e s the r e l a t i ve d i f f e r e n c e s are s m a l l . D i l u t i o n c u r v e s w e r e m a d e o f m u s c l e ex t rac ts so that the a m o u n t o f P V p resen t i n e a c h assay s a m p l e w o u l d f a l l i n the l i n e a r p o r t i o n o f the s t a n d a r d c u r v e . T h e c o n c e n t r a t i o n necessa ry f o r l i n e a r i t y r a n g e d f r o m 1:5 to 1:200 d e p e n d i n g o n m u s c l e t y p e . T a b l e s V I I I a n d I X s u m m a r i z e the P V c o n t e n t i n the v a r i o u s m u s c l e s o f adu l t a n d 2 - w e e k s a m p l e s , r e s p e c t i v e l y . T h e s e va l ues are a lso d e p i c t e d i n h i s t o g r a m s i n F i g u r e s 54 a n d 5 5 . A l l the va lues are e x p r e s s e d as the m e a n P V con ten t . N o r m a l a d u l t E D L m u s c l e s c o n t a i n e d h i g h l eve l s o f P V (27 .89 p g P V / m g T S P ) . T h e a m o u n t o f P V i n the n o r m a l E D L was 5.3 t imes g rea te r t h a n that f o u n d i n the d y s t r o p h i c E D L (5.27 p g P V / m g T S P ) . In con t ras t , v e r y l i t t l e i m m u n o r e a c t i v i t y c o u l d be d e t e c t e d i n the n o r m a l a d u l t so leus m u s c l e ex t rac ts (1.03 p g P V / m g T S P ) , a n d th is v a l u e was 4.5 t i m e s less t h a n the a m o u n t d e t e c t e d i n the d y s t r o p h i c s a m p l e s . O f g rea te r in te res t was the r e s e m b l a n c e o f the P V c o n t e n t o f the a d u l t d y s t r o p h i c E D L w i t h that i n the so leus m u s c l e s . T h i s d r a m a t i c c o n v e r s i o n to s i m i l a r c o n c e n t r a t i o n s o f the p r o t e i n is d e p i c t e d i n F i g u r e 54. A l l o f these r e p o r t e d d i f f e r e n c e s w e r e s i g n i f i c a n t at p <.001. T h e s e resu l ts a re cons i s t en t w i t h the e l e c t r o p h o r e s i s a n d i m m u n o b l o t d a t a , a n d t h e y are i n reasonab le a g r e e m e n t w i t h p r e v i o u s repo r t s o f P V c o n t e n t i n n o r m a l a n d d y s t r o p h i c ske le ta l m u s c l e ( H e i z m a n n et a l . , 1982; Pe t te et a l . , 1985) . D e v e l o p m e n t a l C h a n g e s P a r v a l b u m i n l eve l s w e r e the h ighes t i n the E D L samp les o f the 2 - w e e k - o l d s a m p l e s ( T a b l e I X ) . H o w e v e r , u n l i k e the a d u l t p r e p a r a t i o n s , the m e a n P V c o n t e n t o f the n o r m a l E D L at 2 w e e k s (19.74 p g P V / m g T S P ) was v i r t u a l l y i d e n t i c a l to that o f the d y s t r o p h i c E D L (18.99 p g P V / m g T S P ) as i l l u s t r a t e d i n F i g u r e 55 . T h e s e va lues w e r e s l i g h t l y less t h a n those o b s e r v e d f o r the n o r m a l a d u l t E D L (70 .8% o f the a d u l t 133 F i g u r e 52 S t a n d a r d c u r v e s f o r the r a d i o i m m u n o a s s a y o f ra t s k e l e t a l m u s c l e p a r v a l b u m i n ( o p e n stars) a n d m o u s e m u s c l e h o m o g e n a t e ( so l i d s tars) . T h e m u s c l e h o m o g e n a t e was used at a n i n i t i a l d i l u t i o n o f 1:32 to s i m u l a t e the p u r i f i e d rat p a r v a l b u m i n . V a l u e s f o r p a r v a l b u m i n c o n t e n t i n the 3 2 - w e e k samp les w e r e c a l c u l a t e d f r o m th is c u r v e . 134 i 1 1 1 1 1 1 ! 1 T 0.16 0.31 0.61 1.23 2.3 3.0 10 20 40 80 Parvalbumin ( n g/l00ul) 135" F i g u r e 53 S t a n d a r d c u r v e f o r the r a d i o i m m u n o a s s a y o f ra t s k e l e t a l m u s c l e p a r v a l b u m i n ( b r o k e n l i n e ) a n d m o u s e m u s c l e h o m o g e n a t e ( so l i d l i n e ) . T h e m u s c l e h o m o g e n a t e was used at a n i n i t i a l d i l u t i o n o f 1:32 to s i m u l a t e the p u r i f i e d rat p a r v a l b u m i n . V a l u e s f o r p a r v a l b u m i n c o n t e n t i n the 2 - w e e k samp les w e r e c a l c u l a t e d f r o m th is c u r v e . 136 /37 Figure 54 Histogram of the mean parvalbumin (PV) content determined by RIA, comparing normal (N) and dystrophic (DY) muscle samples from adult EDL and soleus (SOL) muscles. Both the dystrophic EDL and the dystrophic soleus show a dramatic change in mean PV content, approaching similar values to each other. 138 MEAN PV CONTENT (ug/mgTSP) Figure 55 Histogram of the mean parvalbumin (PV) content as determined by R I A , comparing normal (N) and dystrophic (DY) muscle samples from 2-week E D L and soleus (SOL) samples. The amount of P V contained in both soleus samples was beyond the detectable limit of the assay and is therefore expressed as the minimal value detected. Mean parvalbumin content, determined by the R I A , is similar for normal and dystrophic samples at 2 weeks of age. 140 l O S AQ 10S N OO o o o o o o o oo o 103 AQ 103 N g CH m > Z •o < SL 8 m Z 02 v a l u e ) sugges t i ng that syn thes i s o f P V c o m p a r a b l e to a d u l t l eve ls has no t been a t t a i n e d b y 2 w e e k s o f age . T h e R I A f o r the 2 - w e e k samp les was no t sens i t i ve e n o u g h to de tec t s i g n i f i c a n t a m o u n t s o f P V i n e i t he r the n o r m a l o r d y s t r o p h i c so leus p r e p a r a t i o n s . T h e s e q u a n t i t i e s w e r e e x p r e s s e d as the m i n i m u m de tec tab le v a l u e a n d no t the m e a n P V c o n t e n t ( T a b l e I X ) . B a s e d o n these resu l t s , i t w o u l d a p p e a r that pos tna ta l m a t u r a t i o n does no t c o i n c i d e w i t h a s i g n i f i c a n t i nc rease i n P V c o n t e n t i n so leus m u s c l e s . M i n i m a l l eve ls o f P V w e r e de tec ted i n the n o r m a l so leus at b o t h 32 a n d 2 w e e k s o f age . T h e va lues f o r the d y s t r o p h i c so leus at 2 w e e k s o f age w e r e v i r t u a l l y i d e n t i c a l to those o f the n o r m a l so leus ( F i g . 55) . O n l y the d y s t r o p h i c a d u l t so leus s h o w e d a c h a n g e i n m e a n P V c o n t e n t w i t h age. T h e s e resu l ts a re cons i s t en t w i t h those o b t a i n e d b y d i f f e r e n t t e c h n i q u e s to s t u d y the P V c o n c e n t r a t i o n i n e a r l y d e v e l o p m e n t a n d m a t u r a t i o n o f s ke le ta l m u s c l e ( L e b e r e r a n d P e t t e , 1986a ; K l u g et a l . , 1983b ) . I n t e r g r o u p c o m p a r i s o n s o f the E D L m u s c l e s , c o n d u c t e d b y S t u d e n t ' s t - tes t , s h o w e d a s i g n i f i c a n t d i f f e r e n c e (p <.02) b e t w e e n R I A resu l ts o f the 3 2 - w e e k d y s t r o p h i c E D L a n d the 2 - w e e k d y s t r o p h i c E D L . T h e d i f f e r e n c e b e t w e e n n o r m a l 3 2 - w e e k a n d 2 - w e e k E D L assay resu l ts was not s i g n i f i c a n t . T h e r e f o r e , 2 - w e e k E D L m u s c l e s h a v e a l m o s t r e a c h e d t h e i r a d u l t c o n c e n t r a t i o n s o f P V . T h e n o r m a l o r d y s t r o p h i c so leus at 2 w e e k s o f age d i d no t d i f f e r s i g n i f i c a n t l y i n P V c o n t e n t w h e n c o m p a r e d to the n o r m a l a d u l t so leus . In s u m m a r y the m e a n P V c o n t e n t v a r i e d i n a n y g i v e n g r o u p a c c o r d i n g to g e n o t y p e , age a n d m u s c l e t y p e . A l t h o u g h , at 2 w e e k s o f age , g e n o t y p e a n d age h a d less o f a n a f f e c t o n m e a n P V c o n t e n t t h a n d i d m u s c l e t y p e . 142 T A B L E V I I I . M e a n va lues f o r p a r v a l b u m i n c o n t e n t i n 32 w e e k n o r m a l ( N ) a n d d y s t r o p h i c ( D Y ) m u s c l e s a m p l e s . P A R V A L B U M I N C O N T E N T u a / m a T S P u a / m a W E T W T N E D L M E A N 27 .89 1.06 n=6 S . D . 4 .78 0.18 S . E . M . 1.95 0.07 D Y E D L M E A N 5.27 0.15 n=5 S . D . 0 .62 0 .024 S . E . M . 0.28 0.011 N S O L M E A N 1.04 * 0 .032 n=6 S . D . 0.22 0 .009 S . E . M . 0 .09 0 .004 D Y S O L M E A N 4.64 0 .124 n=5 S . D . 0.24 0 .032 S . E . M . 0.11 0 .014 * p <.001 b e t w e e n N a n d D Y g r o u p s f o r b o t h T S P a n d W E T W T . T S P = to ta l s o l u b l e p r o t e i n W E T W T = w e t w e i g h t S . D . = s t a n d a r d d e v i a t i o n S . E . M . = s t a n d a r d e r r o r o f the m e a n 143 T A B L E I X . M e a n va lues f o r p a r v a l b u m i n c o n t e n t i n 2 w e e k n o r m a l ( N ) a n d d y s t r o p h i c ( D Y ) m u s c l e s a m p l e s . P A R V A L B U M I N C O N T E N T p s / m e T S P u e / m e W E T W T N E D L M E A N 19.74 1.031 n=5 S . D . 7.74 0 .582 S . E . M . 3.46 0 .260 D Y E D L M E A N 18.99 0 .622 n=5 S . D . 8.48 0.291 S . E . M . 3.79 0 .130 N S O L L E S S T H A N 1.62 0 .094 n=5 D Y S O L L E S S T H A N 2.33 0 .003 V a l u e s r e p o r t e d f o r the S O L are the m i n i m u m va lues d e t e c t e d m e a n T S P a n d W E T W T . T S P = to ta l s o l u b l e p r o t e i n W E T W T = w e t w e i g h t S . D . = s t a n d a r d d e v i a t i o n S . E . M . = s t a n d a r d e r r o r o f the m e a n 144 D I S C U S S I O N T h i s w o r k represen ts the f i r s t i m m u n o h i s t o c h e m i c a l s t u d y o f P V i n m u r i n e d y s t r o p h i c t i ssue at b o t h p r e c l i n i c a l a n d p o s t c l i n i c a l stages o f the d i sease . It is a lso the f i r s t s t u d y to c o m p a r e the changes i n P V d i s t r i b u t i o n a n d c o n t e n t i n fas t a n d s l o w - t w i t c h s k e l e t a l m u s c l e s o f d y s t r o p h i c m i c e . I m m u n o h i s t o c h e m i s t r v T h e i m m u n o h i s t o c h e m i c a l d i s t r i b u t i o n d a t a o f P V i n n o r m a l a n d d y s t r o p h i c t issue does no t p r o v i d e n e w i n f o r m a t i o n r e g a r d i n g the f u n c t i o n o f P V i n m u s c l e . It i s , h o w e v e r , cons i s ten t w i t h the p r o p o s e d ro le o f P V as a s o l u b l e r e l a x i n g f a c t o r i n f a s t - t w i t c h m u s c l e a n d co r re la tes w e l l w i t h h i s t o c h e m i c a l ( O v a l l e et a l . , 1983) , b i o c h e m i c a l ( Jasch a n d M o a s e , 1985) a n d p h y s i o l o g i c a l (B ress le r et a l . , 1983) da ta . M u s c l e s c o m p o s e d p r i m a r i l y o f f a s t - t w i t c h f i b e r s g e n e r a l l y h a v e a h i g h c o n c e n t r a t i o n o f p a r v a l b u m i n ( C e l i o a n d H e i z m a n n , 1982; H e i z m a n n et a l . , 1982) . T h i s p o r t i o n o f the s t u d y was a n a t t emp t to s i m u l t a n e o u s l y i nves t i ga te the d i s t r i b u t i o n o f p a r v a l b u m i n i n the E D L a n d so leus o f n o r m a l a n d g e n e t i c a l l y d y s t r o p h i c m i c e , a n d the d i s t r i b u t i o n o f e x t r a f u s a l f i b e r s t y p e d a c c o r d i n g to m y o s i n A T P a s e . T h e p resen t d a t a s u p p o r t s these p r e v i o u s repo r t s o n the i m m u n o h i s t o c h e m i c a l d i s t r i b u t i o n o f p a r v a l b u m i n . T h e h i ghes t pe rcen tage o f p a r v a l b u m i n - p o s i t i v e f i b e r s was f o u n d i n t issue sec t i ons f r o m the n o r m a l E D L m u s c l e s ( a p p r o x i m a t e l y 50%) a n d the l owes t i n the so leus ( 8 - 1 0 % ) . T h e r e was a d e f i n i t e p o p u l a t i o n o f d a r k ( p a r v a l b u m i n - p o s i t i v e ) a n d l i g h t ( n o n - r e a c t i v e ) f i b e r s w h i c h mos t l i k e l y c o r r e s p o n d s to t y p e 2 / f a s t - t w i t c h a n d t y p e 1 / s l o w - t w i t c h f i b e r s , r e s p e c t i v e l y . T h o s e m u s c l e ce l l s c l a s s i f i e d as i n t e r m e d i a t e f i b e r s c o m p r i s e d a c o n t i n u u m o f s t a i n i n g i n tens i t i es . T h e s e i n t e r m e d i a t e l y - s t a i n e d f i b e r s p r o b a b l y r ep resen t a s u b g r o u p o f f i b e r s that c a n n o t be c a t e g o r i z e d as e i t he r t y p i c a l t y p e 2 o r t y p e 1 f i b e r s . W h e n the f i b e r n u m b e r s are c o m p a r e d b e t w e e n the p a r v a l b u m i n a n d m y o s i n A T P a s e reac t i ons ( T a b l e s V - V I I ) i t is e v i d e n t that no t a l l 145 the n o n - r e a c t i v e f i b e r s f o r p a r v a l b u m i n are o f the t y p e 1 / s l o w v a r i e t y . T h i s was a lso n o t e d b y C e l i o a n d H e i z m a n n (1982) w h o f o u n d that p a r v a l b u m i n was l o c a l i z e d e x c l u s i v e l y i n the t y p e 2 f i b e r d i s p l a y i n g degrees o f s t a i n i n g i n t e n s i t y . T h e s e i nves t i ga to r s a lso r e p o r t e d that some o f the t y p e 2 A f i b e r s w e r e a c t u a l l y d e v o i d o f p a r v a l b u m i n i m m u n o r e a c t i v i t y a n d r e s e m b l e d the t y p e 1 f i b e r s . T h e r e f o r e , a m u s c l e t y p e d so l e l y a c c o r d i n g to p a r v a l b u m i n i m m u n o r e a c t i v i t y w o u l d be c o n s i d e r e d a s l o w e r m u s c l e t h a n i f t y p e d a c c o r d i n g to m y o s i n A T P a s e r e a c t i v i t y . H e n c e , the resu l ts o f th i s i m m u n o h i s t o c h e m i c a l s t u d y w o u l d suggest that there is no t a t rue c o r r e l a t i o n b e t w e e n p a r v a l b u m i n l o c a l i z a t i o n a n d m y o s i n A T P a s e a c t i v i t y . T h e c o n t i n u u m o f s t a i n i n g i n tens i t i es n o t e d i n P V m a y a lso r e f l e c t the r e c e n t l y d e s c r i b e d he te rogeneous C f i b e r p o p u l a t i o n n o t e d b e t w e e n types 1 a n d 2 A (S ta ron a n d P e t t e , 1986) . T h e s e f i b e r s w e r e c h a r a c t e r i z e d b y the c o - e x i s t e n c e , i n v a r y i n g ra t i os , o f b o t h t y p e 1 s l o w - m y o s i n h e a v y c h a i n s a n d t y p e 2 A f a s t - m y o s i n h e a v y c h a i n s , b u t d i d no t c o r r e l a t e w i t h the m y o s i n l i g h t c h a i n c o m p l e m e n t . I n the a d u l t d y s t r o p h i c t issue the i n c r e a s e d p r o p o r t i o n s o f i n t e r m e d i a t e l y s t a i n e d f i b e r s f o r P V m a y r e f l e c t a g rea te r C f i b e r p o p u l a t i o n . T h i s w o u l d s u p p o r t the h y p o t h e s i s that d y s t r o p h i c m u s c l e e x p e r i e n c e s a l a c k o f d i f f e r e n t i a t i o n as the d isease p rogresses . It is a lso poss ib le that s i n c e no t r ue c o r r e l a t i o n b e t w e e n P V a n d m y o s i n A T P a s e s t a i n i n g was o b s e r v e d i n the p resen t s t u d y , the P V l o c a l i z a t i o n m a y co r re l a te m o r e c l o s e l y w i t h the m y o s i n l i g h t c h a i n c o m p o s i t i o n t h a n the m y o s i n h e a v y c h a i n c o m p l e m e n t o f p a r t i c u l a r f i b e r t ypes . S i n c e P V is a w a t e r - s o l u b l e p r o t e i n , a r t i f a c t u a l r e d i s t r i b u t i o n d u r i n g f i x a t i o n is a l w a y s a p o s s i b i l i t y . A r e d i s t r i b u t i o n m a y be r e s p o n s i b l e f o r the c h e c k e r b o a r d s t a i n i n g p a t t e r n o f the e x t r a f u s a l f i b e r s . T h i s issue has b e e n e x c l u d e d b y s i ng le m u s c l e f i b e r ana l ys i s ( H e i z m a n n et a l . , 1982). T y p e d , s i n g l e m u s c l e f i b e r s f r o m f r e e z e - d r i e d samp les o f ra t m u s c l e w e r e s u b j e c t e d to t w o - d i m e n s i o n a l p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s a n d r e v e a l e d P V to be p resen t o n l y i n the t y p e 2 f i b e r s . In a d d i t i o n , t w o - d i m e n s i o n a l gels o f h e a t - s t a b l e ex t rac ts f r o m h o m o g e n e o u s t y p e 2 B / F G 146 m u s c l e s r e s u l t e d i n v e r y h i g h c o n c e n t r a t i o n s o f P V a f t e r H P L C ana l ys i s o f the same ex t rac t s . T h e s e c o n c e n t r a t i o n s c o r r e l a t e d w e l l w i t h the k n o w n c o n t r a c t i o n a n d h a l f - r e l a x a t i o n t i m e s . I n the p resen t i m m u n o h i s t o c h e m i c a l s t u d y , age was no t a s i g n i f i c a n t f a c t o r i n the d i s t r i b u t i o n o f p a r v a l b u m i n . N o o b v i o u s changes w e r e o b s e r v e d i n the p a r v a l b u m i n i m m u n o s t a i n i n g p a t t e r n f r o m 2 to 32 w e e k s o f age i n the n o r m a l m u s c l e s . A p p a r e n t l y t h e n , the a d u l t p r o f i l e has a lmos t b e e n a t t a i n e d b y 2 w e e k s o f age i n the n o r m a l m o u s e , a n d t h e r e a f t e r , P V l o c a l i z a t i o n va r i es w i t h m u s c l e t y p e a n d no t w i t h age . H o w e v e r , the re was s o m e e v i d e n c e that the 2 - w e e k m u s c l e s are s t i l l o b t a i n i n g m a t u r e c h a r a c t e r i s t i c s i n that the 2 w e e k E D L h a d a s l i g h t l y h i g h e r p e r c e n t a g e o f n o n - r e a c t i v e m u s c l e f i b e r s t h a n i ts a d u l t c o u n t e r p a r t . In a d d i t i o n , the 2 - w e e k so leus s h o w e d a s l i g h t l y h i g h e r p e r c e n t a g e o f P V - p o s i t i v e f i b e r s . In c o n t r a s t , m y o s i n A T P a s e r e a c t i v i t y was a g e - d e p e n d e n t i n b o t h the E D L a n d so leus . T h e 2 - w e e k E D L e x h i b i t e d m o r e s l o w m u s c l e - f i b e r c h a r a c t e r i s t i c s w h e r e a s the so leus m o r e c l o s e l y r e s e m b l e d f a s t - t w i t c h m u s c l e t h a n d i d the 32 w e e k s a m p l e s . T h e r e f o r e , the ra te o f syn thes i s o f P V w o u l d a p p e a r to p l a t e a u b e f o r e the t r a n s i t i o n o f the i s o f o r m s o f m y o s i n has been c o m p l e t e d . T h i s f i n d i n g is cons i s ten t w i t h recen t repo r t s that h a v e s h o w n that changes i n e n z y m e p r o f i l e s , P V , a n d i n the S R o c c u r i n a d v a n c e o f those o b s e r v e d f o r m y o s i n , i n r esponse to c h r o n i c n e r v e s t i m u l a t i o n a n d i n c r e a s e d m u s c u l a r a c t i v i t y ( K l u g et a l . , 1983a ; G r e e n et a l . , 1984) . S i n c e the t r ans i t i ons o f the v a r i o u s sys tems seem to f o l l o w a d i s t i n c t t i m e c o u r s e , P V i m m u n o r e a c t i v i t y m a y be a u s e f u l i n d i c a t o r o f m u s c l e m a t u r i t y . T h e m o s t d r a m a t i c f i n d i n g o b s e r v e d i n th is p o r t i o n o f the s t u d y was the s i m i l a r i t y i n the l o c a l i z a t i o n o f P V i n the a d u l t d y s t r o p h i c E D L w i t h that i n the so leus m u s c l e s . T h e t r e n d t o w a r d a m o r e e v e n d i s t r i b u t i o n was b r o u g h t a b o u t b y a p r e p o n d e r a n c e o f i n t e r m e d i a t e l y - s t a i n e d f i b e r s i n b o t h the fas t a n d s l o w d y s t r o p h i c m u s c l e s . T h i s t r e n d was a lso n o t e d i n the m y o s i n A T P a s e s t a i n i n g p r o f i l e s o f these m u s c l e s . It is l i k e l y that these " i n t e r m e d i a t e " f i b e r s m a y rep resen t no t o n l y t y p e 147 2 / F O G f i b e r s b u t a lso r e g e n e r a t i n g , d e g e n e r a t i n g , o r i m m a t u r e e x t r a f u s a l f i b e r s . T h i s c l a s s i f i c a t i o n o f i n t e r m e d i a t e f i b e r s m a y t h e r e f o r e i n c l u d e those f i b e r s r e f e r r e d to as " a t y p i c a l " , " t r a n s i t i o n a l " , o r " a b n o r m a l " b y o the r i nves t i ga to r s ( D r i b i n a n d S i m p s o n , 1977; O v a l l e et a l . , 1983) . T h i s t r e n d i n the a l t e r a t i o n o f P V - s p e c i f i c p r o p o r t i o n s was no t n o t e d i n the m u s c l e s o f 2 - w e e k - o l d m i c e . O t h e r t h a n s l i gh t d i s t r i b u t i o n v a r i a t i o n s , the l o c a l i z a t i o n o f P V was u n a f f e c t e d b y d y s t r o p h y at th is p r e c l i n i c a l s tage. N o t o n l y was the d i s t r i b u t i o n o f P V s i m i l a r i n n o r m a l a n d d y s t r o p h i c g r o u p s at 2 w e e k s , b u t i t a lso r e s e m b l e d that o f the a d u l t m u s c l e s . T h u s , P V i m m u n o s t a i n i n g appea rs to be e s t a b l i s h e d b y 2 w e e k s o f age i n the m o u s e , a n d i ts d i s t r i b u t i o n is no t a f f e c t e d d u r i n g the i n c i p i e n t stages o f m u r i n e m u s c u l a r d y s t r o p h y . A t least w i t h respec t to P V a n d m y o s i n A T P a s e r e a c t i v i t y , d y s t r o p h y does no t s e e m to a l te r the n o r m a l d e v e l o p m e n t o f m u s c l e ; h o w e v e r , i t m a y d i s t u r b the m a t u r a t i o n p rocess . A l t h o u g h the p resence o f f e t a l m y o s i n ( F i t z s i m o n s a n d H o h , 1983) a n d t y p e 2C f i b e r s ( D u b o w i t z a n d B r o o k e , 1973) i n d y s t r o p h i c m u s c l e m a y i n d i c a t e a f a i l u r e i n c o m p l e t e m u s c l e m a t u r a t i o n o r a r e g r e s s i o n b a c k to a m o r e i m m a t u r e s ta te , P V d i s t r i b u t i o n i n the a d u l t d y s t r o p h i c E D L a n d so leus does no t a d o p t the s t a i n i n g p a t t e r n o b s e r v e d i n the 2 - w e e k - o l d m i c e . H o w e v e r , the c o n c e n t r a t i o n o f P V i n the a d u l t d y s t r o p h i c m u s c l e s m a y i n d i c a t e a m o r e i m m a t u r e state desp i t e the d i f f e r e n c e s i n the s t a i n i n g p r o f i l e f r o m the neona ta l m u s c l e s . T h e i m m u n o h i s t o c h e m i c a l resu l ts suggest that the d y s t r o p h i c m u s c l e s d o no t b e c o m e m o r e f e t a l - l i k e i n t h e i r P V l o c a l i z a t i o n b u t , r a t h e r , s h o w a d i m i n u t i o n i n P V s t a i n i n g d u e to the i n c r e a s e d n u m b e r o f d y s t r o p h i c f i b e r s . It w o u l d be o f in te res t to a s c e r t a i n w h e t h e r those m u s c l e f i b e r s that h a v e b e e n s h o w n to c o n t a i n f e t a l m y o s i n are a lso those tha t s ta i n i n t e r m e d i a t e l y f o r P V , a n d h a v e b e e n d e s c r i b e d as " t r a n s i t i o n a l " o r " a b n o r m a l " b y o the r i n v e s t i g a t o r s . i ) M u s c l e S p i n d l e s B e c a u s e s e r i a l sec t i ons a l o n g the leng ths o f i n d i v i d u a l m u s c l e s p i n d l e s w e r e no t 148 p e r f o r m e d i n the p resen t s t u d y , v e r y l i t t l e c a n be c o n c l u d e d a b o u t the l o c a l i z a t i o n o f P V i n i n t r a f u s a l m u s c l e f i b e r s . It is l i k e l y that r e g i o n a l d i f f e r e n c e s ex i s t i n the d i s t r i b u t i o n o f P V i n i n t r a f u s a l f i b e r s , s i m i l a r to those n o t e d i n m y o s i n A T P a s e r e a c t i v i t y as d i s c u s s e d i n C h a p t e r 1. R e g a r d l e s s o f g e n o t y p e , i n p o l a r r eg ions o f m u s c l e s p i n d l e s sec t i ons o f a d u l t m u s c l e s o c c a s i o n a l l y c o n t a i n e d c h a i n f i b e r s that w e r e p o s i t i v e l y s t a i n e d f o r P V . O n the o t h e r h a n d , c h a i n a n d b a g f i b e r s i n the 2 - w e e k m u s c l e s o f b o t h n o r m a l a n d d y s t r o p h i c m i c e w e r e i m m u n o r e a c t i v e i n p o l a r r e g i o n s . N o s t a i n i n g o f the i n t r a f u s a l f i b e r s , h o w e v e r , was o b s e r v e d i n e q u a t o r i a l a n d j u x t a e q u a t o r i a l zones o f m u s c l e s p i n d l e s i n e i t he r o f the age g r o u p s . S i n c e mos t o f the c o n t r a c t i l e a p p a r a t u s o f an i n t r a f u s a l f i b e r is f o u n d w i t h i n the p o l a r r e g i o n o f the s p i n d l e i t m a y no t be s u r p r i s i n g that P V w o u l d a lso be c o n c e n t r a t e d h e r e . H o w e v e r , i f P V is a t r u l y c y t o p l a s m i c p r o t e i n w i t h no a f f i n i t y f o r i n t r a c e l l u l a r s t r u c t u r e s , th is resu l t m a y rep resen t e v i d e n c e f o r some p a r t i c u l a r i n t r a c e l l u l a r l o c a l i z a t i o n . P V i m m u n o r e a c t i v i t y has b e e n r e p o r t e d i n one o f the c h a i n f i b e r s o f m u s c l e s p i n d l e s i n a d u l t ra t E D L a n d so leus m u s c l e s b u t no t i n the n u c l e a r b a g f i b e r s ( C e l i o a n d H e i z m a n n , 1982) . T h e s e i nves t i ga to r s r e p o r t e d that the i m m u n o s t a i n i n g was u n e q u i v o c a l i n s p i n d l e s o f the so leus bu t no t i n those o f the E D L . It is k n o w n that m a m m a l i a n c h a i n f i b e r s h a v e fas te r r e l a x a t i o n p r o p e r t i e s t h a n b a g f i b e r s ( B o y d a n d S m i t h , 1984) A l t h o u g h th is m a y a c c o u n t f o r the p o s i t i v e r e a c t i o n o f the c h a i n f i b e r to P V ( C e l i o a n d H e i z m a n n , 1982) , i t is o f i n te res t that o n l y one o f the c h a i n f i b e r s s h o w s i m m u n o r e a c t i v i t y . M y o s i n A T P a s e r e a c t i v i t y a p p e a r e d i d e n t i c a l i n the t w o i n t r a f u s a l c h a i n f i b e r s as d e s c r i b e d i n C h a p t e r 1. A s i n the e x t r a f u s a l f i b e r s , i t w o u l d a p p e a r that n e i t h e r P V i m m u n o r e a c t i v i t y n o r m y o s i n A T P a s e s t a i n i n g c a n be used sepa ra te l y as a m e a n s o f d e t e r m i n i n g i n t r a f u s a l f i b e r t ypes s i n c e there is no t rue c o r r e l a t i o n b e t w e e n the t w o . T h e p r e s e n c e o f P V i n n u c l e a r b a g f i b e r s o f m u s c l e s p i n d l e s i n the 2 - w e e k m u s c l e s m a y be a s i g n o f i m m a t u r i t y i n the syn thes i s o f th is p r o t e i n . H o w e v e r , the f a c t that the syn thes i s o f P V g e n e r a l l y p r o c e e d s that o f m y o s i n i s o f o r m s , a n d that 149 a d u l t m y o s i n A T P a s e s t a i n i n g pa t te rns are e s t a b l i s h e d b y 2 w e e k s o f age i n m u r i n e i n t r a f u s a l f i b e r s (see C h a p t e r 1), i t is u n l i k e l y that P V - p o s i t i v e r e a c t i v i t y o f bag f i b e r s is a d e v e l o p m e n t a l p h e n o m e n o n . B a g 2 i n t r a f u s a l f i b e r s h a v e b e e n d e s c r i b e d as fas t n u c l e a r b a g f i b e r s d u e to t he i r r a p i d c o n t r a c t i o n a n d r e l a x a t i o n t imes w h e n c o m p a r e d to b a g l f i b e r s ( B o y d a n d S m i t h , 1984) . It is p o s s i b l e tha t the u n i q u e p h y s i o l o g i c a l p r o p e r t i e s o f i n t r a f u s a l f i b e r s m a y a lso co r re l a te the i m m u n o l a b e l l i n g o b s e r v e d i n th is s t u d y . W h y P V was no t o b s e r v e d i n b a g f i b e r s o f the a d u l t musc les is u n k n o w n . P a r v a l b u m i n C o n t e n t A l t h o u g h i m m u n o c y t o c h e m i c a l s t a i n i n g p r o c e d u r e s m a y s p e c i f i c a l l y l o c a l i z e P V i n a p a r t i c u l a r c e l l , t h e y d o no t p r o v i d e v e r y q u a n t i t a t i v e i n f o r m a t i o n . D e t e r m i n i n g the c e l l u l a r c o n t e n t o f th is p r o t e i n b y the i n t e n s i t y o f the s t a i n is a v e r y s u b j e c t i v e e x e r c i s e . T h e i m m u n o h i s t o c h e m i c a l resu l ts i n th is s t u d y r e v e a l e d d r a m a t i c d i f f e r e n c e s i n P V d i s t r i b u t i o n no t o n l y b e t w e e n m u s c l e s , b u t a lso b e t w e e n i n d i v i d u a l f i b e r t ypes a n d age g r o u p s . M o r e n o t e w o r t h y w e r e the a l t e ra t i ons i n the l o c a l i z a t i o n o f P V i n the d y s t r o p h i c s a m p l e s . It was t h e r e f o r e o f in te res t to q u a n t i f y these changes a n d t h e r e b y d e t e r m i n e w h e t h e r the i m m u n o h i s t o c h e m i c a l resu l ts c o u l d be subs tan t i a ted b i o c h e m i c a l l y . T h e b i o c h e m i c a l f i n d i n g s p resen ted he re s u p p o r t the resu l ts o f the p r e c e d i n g i m m u n o h i s t o c h e m i c a l e x p e r i m e n t s . T h e p resence o f P V , as d e t e r m i n e d b y ge l e l e c t r o p h o r e s i s a n d i m m u n o b l o t t i n g , a n d the P V c o n t e n t d e t e r m i n e d b y R I A w e r e c o n s i s t e n t l y s h o w n to be m o r e p r e v a l e n t i n the E D L t h a n i n the so leus . T h i s is t rue f o r the t i ssue s a m p l e d at b o t h 2 a n d 32 w e e k s o f age i n the n o r m a l p o p u l a t i o n , a n d i n the 2 - w e e k d y s t r o p h i c age g r o u p . P a r v a l b u m i n va lues w e r e a p p r o x i m a t e l y 27 t imes g rea te r i n the a d u l t E D L t h a n i n the a d u l t so l eus , a n d a p p r o x i m a t e l y 12 t i m e s grea ter i n the E D L t h a n i n the so leus o f the y o u n g e r a n i m a l s . T h i s d i f f e r e n c e i n the c o n c e n t r a t i o n o f P V b e t w e e n f a s t - t w i t c h a n d s l o w - t w i t c h m u s c l e s is cons i s ten t w i t h 150 s i m i l a r t rends r e p o r t e d p r e v i o u s l y i n o the r spec ies ( L e b e r e r a n d P e t t e , 1986a ,b ; H e i z m a n n , 1984; H e i z m a n n et a l . , 1982) . C o l l e c t i v e l y , these resu l ts s u p p o r t the h y p o t h e s i s that P V c o n t e n t is f i b e r t y p e s p e c i f i c ( C e l i o a n d H e i z m a n n , 1982) , i n that m u s c l e s c o m p o s e d p r e d o m i n a n t l y o f t ype 2 f i b e r s h a v e a h i g h e r P V c o n c e n t r a t i o n t h a n m u s c l e s that a re p r i m a r i l y t y p e 1 i n c o m p o s i t i o n . T h e s e n s i t i v i t y o f r a d i o i m m u n o a s s a y d e p e n d s o n the s p e c i f i c i t y o f the a n t i s e r u m , o p t i m a l d i l u t i o n ra t ios o f a n t i b o d y a n d a n t i g e n , a n d the p u r i t y o f the a n t i g e n used f o r the i o d i n a t i o n ( C o o p e r , 1979) . It s h o u l d be r e m e m b e r e d that the i m m u n o c h e m i c a l a p p r o a c h is a n i n d i r e c t one i n w h i c h the o b s e r v e d c o m p e t i t i o n m a y be the resu l t o f a s m a l l a m o u n t o f c o m p e t i t o r w h o s e i d e n t i t y is the same as the s t a n d a r d a n t i g e n , o r a l a rge r q u a n t i t y o f m a t e r i a l that has a s i m i l a r a n t i g e n i c d e t e r m i n a n t ( C o o p e r , 1979) . T h e P V va l ues o b t a i n e d b y the R I A i n the p resen t s t u d y are l o w c o m p a r e d to p r e v i o u s l y p u b l i s h e d repor t s ( H e i z m a n n et a l . , 1982; K l u g et a l . , 1985 ; E n d o et a l . , 1986b) . A p o s s i b l e s o u r c e f o r th is d i s c r e p a n c y is the f a c t that the a n t i s e r u m h a d a l o w t i t re o f 1 :400 -500 . T h e a f f i n i t y o f the a n t i s e r a m a y h a v e b e e n m a s k e d b y the p r e s e n c e o f v a r i o u s o t h e r a n t i b o d i e s i n the s e r u m . A s w e l l , H e i z m a n n heat t reats h is s a m p l e s s u c h that the T S P va lues are l o w e r t h a n ou rs a n d the T P o v e r T S P va lues are g rea te r t h a n o u r s . T h e i m m u n o h i s t o c h e m i c a l d a t a s h o w e d that the o v e r a l l d i s t r i b u t i o n o f P V i n the 2 - w e e k n o r m a l s was no t s i g n i f i c a n t l y d i f f e r e n t f r o m that o f the a d u l t m u s c l e s . T h e c o n c e n t r a t i o n o f P V i n the E D L , h o w e v e r , is age d e p e n d e n t . B y 2 w e e k s o f age the P V c o n t e n t is 7 0 % o f tha t f o u n d i n the n o r m a l a d u l t E D L . So leus c o n c e n t r a t i o n s b e t w e e n age g r o u p s w e r e v i r t u a l l y i d e n t i c a l to e a c h o the r . T h e r e f o r e , a l t h o u g h the p a t t e r n o f P V d i s t r i b u t i o n has b e e n e s t a b l i s h e d b y 2 w e e k s o f age , the syn thes i s o f P V w i t h i n the p o s i t i v e l y - s t a i n e d f i b e r s has no t r e a c h e d i ts p e a k . T h e p resen t f i n d i n g s are i n a g r e e m e n t w i t h p r e v i o u s d e v e l o p m e n t a l s tud ies that h a v e d e t e c t e d P V b y pos tna ta l d a y 4 o r 5 i n the E D L o f the rat ( C e l i o a n d H e i z m a n n , 1982; B e r c h t o l d a n d M e a n s , 1985; L e b e r e r a n d P e t t e , 1986b) . I n o n e - w e e k - o l d m i c e , 151 the P V c o n t e n t rep resen ts 5 - 1 3 % o f that f o u n d i n the a d u l t ( K l u g et a l . , 1985) . T h e i n i t i a l s yn thes i s a n d t i m e cou rse o f the i nc reases i n P V a p p e a r to o c c u r a s y n c h r o n o u s l y i n v a r i o u s f a s t - t w i t c h m u s c l e s ( L e b e r e r a n d P e t t e , 1986b ; L e P e u c h et a l . , 1979) . W h i l e m a t u r e l eve l s o f P V h a v e b e e n n o t e d i n the g a s t r o c n e m i u s a n d psoas J o f the r a b b i t b y 9 w e e k s a f t e r b i r t h , s i m i l a r l eve ls i n the E D L h a v e no t b e e n o b t a i n e d a f t e r 17 w e e k s ( L e b e r e r a n d P e t t e , 1986b) . B y 8 - 1 2 w e e k s o f age , a d u l t l eve ls o f P V i n the E D L o f the m o u s e h a v e b e e n a c h i e v e d ( J a s c h et a l . , 1982 , J a s c h a n d M o a s e , 1985 ; K l u g et a l . , 1985) . I n a m o r e recen t d e v e l o p m e n t a l s t u d y o f the rat g a s t r o c n e m i u s m u s c l e , P V m R N A was f i r s t d e t e c t e d at d a y 5 p o s t n a t a l l y , a n d s u b s e q u e n t l y i n c r e a s e d 15 to 2 0 - f o l d b y d a y 20 to a c h i e v e i ts m a x i m u m leve ls ( B e r c h t o l d a n d M e a n s , 1985) . In the c h i c k e m b r y o , o t h e r i n v e s t i g a t o r s h a v e s h o w n that a c t i v e syn thes i s o f P V beg ins 2 days b e f o r e h a t c h i n g i n m u s c l e s o f the l e g , f o l l o w e d b y i ts syn thes i s i n the pec to ra l i s m u s c l e 4 - 5 d a y s a f t e r h a t c h i n g , r e a c h i n g m a x i m a l l eve l s b y d a y 7 ( L e P e u c h et a l . , 1979) . R e s u l t s f r o m these s t u d i e s , t h e r e f o r e , i n d i c a t e that P V syn thes i s i n ske le ta l m u s c l e s is d e v e l o p m e n t a l l y r e g u l a t e d . T h e d e v e l o p m e n t a l r e g u l a t i o n o f P V i n these v a r i o u s m u s c l e s appea rs to co r re l a te w i t h the d i f f e r e n t i a t i o n p rocesses w h i c h l e a d to the f o r m a t i o n o f f a s t - c o n t r a c t i n g a n d r a p i d l y - r e l a x i n g e x t r a f u s a l m u s c l e f i b e r s , ( B e r c h t o l d a n d M e a n s , 1985; H e i z m a n n , 1984) . T h e s e i n c l u d e a dec rease i n r e l a x a t i o n t i m e a f t e r a t w i t c h o r t e tanus , a n d the syn thes i s o f e l e m e n t s o f the c a l c i u m c y c l e , i n p a r t i c u l a r the S R c a l c i u m p u m p a n d m y o s i n l i g h t c h a i n k i n a s e ( L e P e u c h et a l . , 1979) . T h i s has r e c e n t l y b e e n s u p p o r t e d b y a s t u d y d e s i g n e d to see i f P V is i n v o l v e d i n the ro le o f t issue d i f f e r e n t i a t i o n , c e l l m i g r a t i o n a n d the even ts o f e a r l y e m b r y o d e v e l o p m e n t ( K a y et a l . , 1987) . U s i n g i so la ted c D N A segmen ts e n c o d i n g a P V o f t a d p o l e s , a n d a v a i l a b l e a n t i b o d i e s as m o l e c u l a r p r o b e s i t was f o u n d that P V a n d i ts m R N A is absen t d u r i n g ea r l y - d e v e l o p m e n t . T h e f i r s t d e m o n s t r a b l e a p p e a r a n c e o f P V o c c u r r e d at the t i m e o f som i t e d i f f e r e n t i a t i o n w h e n m u s c l e ce l l s w e r e a c t i v e i n e x p r e s s i o n o f the c o n t r a c t i l e a p p a r a t u s . It was sugges ted that the c o o r d i n a t e d e x p r e s s i o n o f P V a n d c o n t r a c t i l e 152 p r o t e i n s a lso s u p p o r t s the b e l i e f that P V p l a y s a ro le i n the c o n t r a c t i o n - r e l a x a t i o n even ts o f s ke le ta l m u s c l e . T h e b i o c h e m i c a l da ta i n the p resen t s t u d y ( T a b l e V I I I ) s h o w e d that the a d u l t d y s t r o p h i c E D L c o n t a i n s s i g n i f i c a n t l y less P V t h a n the n o r m a l E D L , cons i s t en t w i t h the o b s e r v e d i m m u n o h i s t o c h e m i c a l c h a n g e s . A n 8 1 % d i m i n u t i o n f r o m n o r m a l va lues was n o t e d i n the 32 w e e k d y s t r o p h i c E D L . T h i s r e d u c t i o n is l a r g e r t h a n the 4 0 % dec rease i n P V r e p o r t e d p r e v i o u s l y f o r v a r i o u s f a s t - t w i t c h d y s t r o p h i c m u s c l e s at 15 w e e k s o f age ( K l u g et a l . , 1985) . B o t h o f these f i n d i n g s a r e , h o w e v e r , cons i s t en t w i t h p u b l i s h e d d a t a o n c h a n g e s i n p r o t e i n d i s t r i b u t i o n s i n m o u s e m u s c l e d u r i n g p rog ress i ve pos tna ta l stages o f the d isease ( Jasch et a l . , 1982) . N o r m a l i z e d d a t a f r o m scans o f these I E F gels i n d i c a t e that there is a g r a d u a l d e p l e t i o n o f P V f r o m 12 to 32 w e e k s o f age i n the E D L o f the d y s t r o p h i c m o u s e . T h e r e f o r e , the 8 1 % d r o p n o t e d i n the p resen t s t u d y is p r o b a b l y d u e to the a d v a n c e d stage o f the d isease r e s u l t i n g f r o m a d e g r a d a t i o n o f P V as the e x p r e s s i o n o f e x t r a f u s a l f i b e r t ypes c h a n g e . O n the o t h e r h a n d , a s i m i l a r t r e n d was no t o b s e r v e d i n the so leus m u s c l e s . In f a c t , the c o n c e n t r a t i o n o f P V i n the a d u l t d y s t r o p h i c so leus s u r p a s s e d that o f the n o r m a l so leus to l eve l s a p p r o a c h i n g those o f the d y s t r o p h i c E D L . H e n c e , these resu l ts s u p p o r t o u r d a t a o n the i m m u n o h i s t o c h e m i c a l d i s t r i b u t i o n o f P V i n the fas t a n d s l o w m u s c l e s o f the d y s t r o p h i c a n i m a l s . It w o u l d a p p e a r , t h e n , tha t the E D L a n d so leus are b e c o m i n g m o r e a l i k e w i t h respec t to c o n t e n t a n d d i s t r i b u t i o n o f P V , as w e l l as i n t h e i r p a t t e r n o f s o l u b l e m u s c l e p r o t e i n s as n o t e d i n the ge ls . T h e resu l ts f r o m o u r e x p e r i m e n t s o n the n o r m a l so leus m u s c l e s w e r e a lso n o t e w o r t h y . P a r v a l b u m i n was u n d e t e c t a b l e b y g e l e l e c t r o p h o r e s i s , b a r e l y e v i d e n t o n the i m m u n o b l o t s , a n d o n l y m i n i m a l va lues c o u l d be o b t a i n e d w i t h the R I A . H o w e v e r , the i m m u n o h i s t o c h e m i c a l d a t a i n d i c a t e d tha t , t a k e n c o l l e c t i v e l y , 2 2 % o f the e x t r a f u s a l f i b e r p o p u l a t i o n i n the n o r m a l so leus c o n t a i n e d s o m e l e v e l o f P V . A poss ib le e x p l a n a t i o n f o r th is m a y l i e i n the f a c t that a l l the b i o c h e m i c a l d a t a was c o l l e c t e d o n w h o l e m u s c l e h o m o g e n a t e s a n d no t f r o m s i ng l e f i b e r s , w h e r e a s the 153 i m m u n o h i s t o c h e m i c a l s t a i n i n g i d e n t i f i e d P V i n sec t i ons o f i n d i v i d u a l m u s c l e ce l l s . In a d d i t i o n , e v e n t h o u g h there is a r e l a t i v e l y h i g h p e r c e n t a g e o f p o s i t i v e f i b e r s i n d i c a t i n g the p r e s e n c e o f P V , the a c t u a l P V c o n c e n t r a t i o n w i t h i n these f i b e r s m a y no t be i n s u f f i c i e n t a m o u n t s to be d e t e c t e d b y g e l e l e c t r o p h o r e s i s o r R I A . It is o b v i o u s that m u s c u l a r d y s t r o p h y does no t a f f e c t the c o n c e n t r a t i o n o f P V d u r i n g the p r e c l i n i c a l stages o f the d isease i n the d y 2 J m o u s e (see T a b l e I X ) . A n o t e w o r t h y f e a t u r e emerges w h e n a c o m p a r i s o n o f the p r e c l i n i c a l a n d p o s t c l i n i c a l va l ues f o r b o t h the d y s t r o p h i c E D L a n d so leus m u s c l e s are m a d e . T h e c o n t e n t o f P V i n the a d u l t E D L has d i m i n i s h e d b e y o n d that o f the 2 - w e e k E D L , w h e r e a s the va lues f o r the so leus h a v e i n c r e a s e d s i g n i f i c a n t l y a b o v e those o f the 2 - w e e k m u s c l e . T h e s e f i n d i n g s suggest that the t w o m u s c l e s are a d a p t i n g to a n i n t e r m e d i a t e f o r m that m o r e c l o s e l y r esemb les the m u s c l e s i n the p r e c l i n i c a l stages o f d y s t r o p h y . T h e s e a l te ra t ions i n P V s u p p o r t the h y p o t h e s i s that d y s t r o p h y m a y r e f l e c t a n i m m a t u r e state o r an a l t e r a t i o n i n the d i f f e r e n t i a t i o n p rocess w i t h respec t to the c a l c i u m r e g u l a t o r y s y s t e m ( K l u g et a l . , 1985; Pe t te et a l . , 1985) . A s i l l u s t r a t e d i n F i g u r e 3 4 , P V was not the o n l y s o l u b l e m u s c l e p r o t e i n that was a l t e red b y m u r i n e d y s t r o p h y . O f p a r t i c u l a r in te res t a re a n u m b e r o f b a n d s i n the 1 6 - 2 0 K D r a n g e . T h o s e b a n d s c h a r a c t e r i s t i c o f the s l o w m u s c l e s n o w a p p e a r i n the d y s t r o p h i c fas t m u s c l e . A r e c i p r o c a l p h e n o m e n o n o c c u r s i n the d y s t r o p h i c so leus . S o m e , o r a l l , o f these h i g h e r m o l e c u l a r - w e i g h t p r o t e i n s m a y a lso be e i t he r c a l c i u m - b i n d i n g o r c a l c i u m - r e g u l a t e d p r o t e i n s . A c c o r d i n g to the p o s i t i o n o f these b a n d s i t is q u i t e p r o b a b l e that t hey rep resen t the v a r i o u s m y o s i n l i g h t c h a i n s ( L C I - f a s t , 22 .5 K D ; L C 2 - f a s t , 18 K D ; L C 3 - f a s t , 16.5 K D ; L C I - s l o w , 27 K D ; L C 2 - s l o w , 19 K D ) , t r o p o n i n - C , 18 K D a n d c a l m o d u l i n , 16.7 K D . A n a l t e r a t i o n i n the m y o s i n L C c o m p o s i t i o n i n m u r i n e d y s t r o p h y m a y a lso r e f l e c t a loss o f the n o r m a l d i f f e r e n t i a t i o n p rocess . It is n o t e w o r t h y that no t a l l the m y o s i n L C ' s are s i m i l a r l y a f f e c t e d i n m u r i n e d y s t r o p h y ( F i t z s i m o n s a n d H o h , 1983 ; J a s c h a n d M o a s e , 1985) . It is no t k n o w n w h a t the i n t e r r e l a t i o n s h i p is b e t w e e n these p r o t e i n s a n d P V . F u r t h e r s p e c u l a t i o n s h o u l d not be 154 m a d e u n t i l the i d e n t i t y o f these p r o t e i n s is k n o w n . A c r i t i c a l f o l l o w - u p to the p resen t s t u d y w o u l d be to d e t e r m i n e w h e t h e r the o t h e r m a j o r c a l c i u m - b i n d i n g p r o t e i n s , n a m e l y c a l m o d u l i n a n d t r o p o n i n - C , are a f f e c t e d i n a s i m i l a r f a s h i o n b y the d y s t r o p h i c p rocess . B e s i d e s the v a r i o u s e n z y m e a c t i v a t o r a c t i v i t i e s o f c a l m o d u l i n , s u c h as the p h y s i o l o g i c a l C a 2 + - d e p e n d e n t r e g u l a t i o n o f m y o s i n l i g h t c h a i n k i n a s e i n a l l three m u s c l e t ypes ( W a l s h et a l . , 1980) , c a l m o d u l i n c a n e f f e c t i v e l y subs t i t u te f o r t r o p o n i n - C i n r e s t o r i n g C a 2 + - s e n s i t i v i t y to a c t o m y o s i n A T P a s e . A n d , a l t h o u g h c a l m o d u l i n does no t s t i m u l a t e C a 2 + - u p t a k e i n the S R o f e i t h e r fas t o r s l o w m u s c l e ( E i b s c h u t z et a l . , 1984) c a l m o d u l i n m a y be i n v o l v e d i n C a 2 + - r e l e a s e f r o m ske le ta l S R ( C h i e s i a n d C a r a f o l i , 1982). A l t h o u g h the ro le o f c a l m o d u l i n i n the r e g u l a t i o n o f s k e l e t a l m u s c l e S R a n d C a 2 + - t r a n s p o r t r e m a i n s to be d e t e r m i n e d , i t appea rs that th is p r o t e i n m a y be i n v o l v e d i n the c a l c i u m - r e g u l a t i o n o f m u s c l e c o n t r a c t i o n . H o w e v e r , i t is poss ib le c a l m o d u l i n c o u l d a lso subs t i t u te f o r P V i n the r e l a x a t i o n phase o f the c y c l e . I f the f u n c t i o n o f c a l m o d u l i n is a l t e red i n d y s t r o p h y th is m a y a lso c o n t r i b u t e to the changes i n the p h y s i o l o g i c a l response o f m u s c l e i n m u r i n e d y s t r o p h y . E x t r a c t s f r o m d y s t r o p h i c c h i c k e n m u s c l e p r e t r e a t e d w i t h c a l c i u m - a c t i v a t e d n e u t r a l p ro tease e x h i b i t a dec rease i n t r o p o n i n - I a n d t r o p o n i n - C w i t h a r e l a t i ve p r e s e r v a t i o n o f t r o p o n i n - T (Sug i t a et a l . , 1980) . S tud ies o f s k e l e t a l m u s c l e f r o m hams te rs h a v e d e m o n s t r a t e d no de tec tab le d i f f e r e n c e s b e t w e e n the c a l m o d u l i n con ten t o f n o r m a l a n d d y s t r o p h i c a n i m a l s ; t h e r e f o r e , n o a p p a r e n t c o r r e l a t i o n ex i s t s b e t w e e n the c a l c i u m a c c u m u l a t i o n o b s e r v e d i n d y s t r o p h i c m u s c l e a n d the r e c o r d e d changes i n c a l m o d u l i n c o n t e n t ( K l a m u t et a l . , 1983) . A l t h o u g h the dec rease i n m u s c l e p r o t e i n s i n d y s t r o p h y is l a r g e l y a t t r i b u t e d to an i n c r e a s e d rate o f p r o t e i n d e g r a d a t i o n ( G a r b e r et a l . , 1980) , i t appea rs tha t , desp i te c lose s i m i l a r i t i e s i n p r o t e i n s s u c h as c a l m o d u l i n , t r o p o n i n - C a n d P V , e a c h one m a y be a f f e c t e d d i f f e r e n t l y b y the same d isease . In m u r i n e d y s t r o p h y , the a l te ra t ions o b s e r v e d i n P V c o n t e n t are l a r g e l y d u e to a c o n v e r s i o n i n the p h e n o t y p i c e x p r e s s i o n 155 of extrafusal fiber types. This conversion to an intermediate type or perhaps an immature state may be a consequence of the degradation process triggered by a defect in calcium metabolism. 156 G E N E R A L D I S C U S S I O N It was no t u n e x p e c t e d that no a l t e ra t i ons w e r e o b s e r v e d i n the neona ta l i n t r a f u s a l f i b e r s a n d m u s c l e s p i n d l e s i n the d y s t r o p h i c m i c e . H o w e v e r , th is o b s e r v a t i o n is i n i t se l f i n t e r e s t i n g w h e n one c o n s i d e r s the c o m p l e x i n n e r v a t i o n pa t t e rn to these s e n s o r y r ecep to r s a n d the a l t e ra t i ons w h i c h h a v e b e e n d e s c r i b e d i n b o t h the senso ry a n d m o t o r n e r v e s u p p l y to d y s t r o p h i c m u s c l e . It is p o s s i b l e that c o m p l e x i t y o f the m u s c l e s p i n d l e s p o l y n e u r o n a l i n n e r v a t i o n is i ts o w n s a f e g u a r d m e c h a n i s m . It is a lso t e m p t i n g to specu la te that the i n t r a f u s a l f i b e r m a y be g o v e r n e d b y n e u r o t r o p h i c i n f l u e n c e s w h i c h are d i f f e r e n t f r o m those o f i ts n e i g h b o u r i n g e x t r a f u s a l f i b e r s , a n d w h i c h are s p a r e d the d y s t r o p h i c gene e x p r e s s i o n . A s w e l l , the a c t u a l c o m b i n a t i o n o r c o m p o s i t i o n o f v a r i o u s m y o s i n h e a v y c h a i n s a n d m y o s i n l i g h t c h a i n s w i t h i n the i n t r a f u s a l f i b e r s m a y i n f a c t m a s k a n y h i s t o c h e m i c a l changes w h i c h m a y o c c u r as a resu l t o f the d i sease . A l t h o u g h P V m a y be a u s e f u l i n d i c a t o r o f a m u s c l e ' s state o f m a t u r i t y , a n d p e r h a p s a m a r k e r f o r e a r l y n e u r o m u s c u l a r d i s t u r b a n c e s , s i g n i f i c a n t changes i n P V l o c a l i z a t i o n c o u l d no t be d e t e c t e d i n the i n t r a f u s a l f i b e r s . H o w e v e r , w i t h o u t a m o r e r i g o r o u s i n v e s t i g a t i o n i n t o the a c t u a l d i s t r i b u t i o n o f P V t h r o u g h o u t the en t i r e l e n g t h o f the i n t r a f u s a l f i b e r s , n o t h i n g c o n c l u s i v e c a n be s a i d a b o u t i ts a p p e a r a n c e i n d y s t r o p h y w i t h rega rds to the s p i n d l e . T h e d a t a p r e s e n t e d i n C h a p t e r 2 o f th is thes is i n d i c a t e s that a dec rease i n p a r v a l b u m i n c o n t e n t o c c u r s i n the e x t r a f u s a l f i b e r s o f the d y s t r o p h i c E D L w h e r e a s an i nc rease i n p a r v a l b u m i n c o n t e n t o c c u r s i n the d y s t r o p h i c so leus w i t h age . T h e s e a l te ra t i ons are cons i s t en t w i t h the k n o w n changes that o c c u r i n the f a s t - t w i t c h m u s c l e s i n h a l f - r e l a x a t i o n t i m e s . T h i s s t u d y a lso p r o v i d e s f u r t h e r i n d i r e c t e v i d e n c e f o r the p r o p o s e d ro le o f p a r v a l b u m i n as a s o l u b l e r e l a x a t i o n f a c t o r i n f a s t - t w i t c h s k e l e t a l m u s c l e . In b o t h the d y s t r o p h i c E D L a n d so leus there appea rs to be a c o n v e r s i o n o f 157 e x t r a f u s a l f i b e r t ypes to a m o r e i n t e r m e d i a t e f o r m t h a n tha t seen i n n o r m a l a g e - m a t c h e d m u s c l e s . T h i s i n t e r m e d i a t e f o r m o f m y o f i b e r does no t c o m p l e t e l y r e s e m b l e n e o n a t a l m u s c l e s at 2 w e e k s o f age i n e i t h e r the c o n t e n t o r the d i s t r i b u t i o n o f p a r v a l b u m i n . T h e c o n c e n t r a t i o n o f p a r v a l b u m i n f o u n d i n these d y s t r o p h i c musc les d o e s , h o w e v e r , r e s e m b l e the leve ls r e p o r t e d i n m o r e i m m a t u r e m u s c l e . T h e s l i gh t i n c r e a s e i n p a r v a l b u m i n i n the a d u l t d y s t r o p h i c so leus a n d the a p p e a r a n c e o f h i g h e r m o l e c u l a r - w e i g h t p r o t e i n b a n d s i n the d y s t r o p h i c E D L r e s e m b l i n g those o b s e r v e d i n the s l o w - t w i t c h m u s c l e , a lso s u p p o r t the h y p o t h e s i s that d y s t r o p h y i n s o m e w a y a l ters the d i f f e r e n t i a t i o n p rocess a n d the subsequen t m a t u r a t i o n o f r e g e n e r a t i n g f i b e r s . T h e changes o b s e r v e d i n th i s d isease i n b o t h p a r v a l b u m i n c o n t e n t a n d the d i s t r i b u t i o n o f f i b e r t ypes m a y r e f l e c t a n i m m a t u r e state o f d y s t r o p h i c m u s c l e . T h i s is cons i s ten t w i t h the r e p o r t e d c o n v e r s i o n s i n e x t r a f u s a l f i b e r t y p e s , i s o m y o s i n s a n d C a 2 + m e t a b o l i s m to a neona ta l s tage o f d e v e l o p m e n t . I n t e r e s t i n g l y , p a r v a l b u m i n m a t u r a t i o n pa ra l l e l s that o f the S R a n d o t h e r c a l c i u m - r e g u l a t e d en t i t i es . M u s c u l a r d y s t r o p h y , t h e r e f o r e , appea rs to i n f l u e n c e the m a t u r a t i o n o r d i f f e r e n t i a t i o n o f n o r m a l fas t m u s c l e f i b e r s , p a r t i c u l a r l y the e l emen ts i n v o l v e d i n the C a 2 + c y c l e . T h e r e are s e v e r a l i n d i c a t i o n s that ske le ta l m u s c l e m a t u r a t i o n is p r o l o n g e d o r a l t e red i n m u r i n e d y s t r o p h y . N o r m a l d i f f e r e n t i a t i o n o f fas t m u s c l e i n c l u d e s the w i t h d r a w a l o f p o l y n e u r o n a l i n n e r v a t i o n , the s w i t c h i n g o f f o f s l o w - t w i t c h m u s c l e m y o s i n , a n i n c r e a s e i n the a c t i v i t y o f g l y c o l y t i c e n z y m e s a n d the u p t a k e - c a p a c i t y o f the S R f o r C a 2 + , a n d a dec rease i n c o n t r a c t i o n a n d h a l f - r e l a x a t i o n t i m e s ( V r b o v a , 1983 ; D h o o t , 1985) . S y n t h e s i s o f these e lemen ts that are pa r t o f the C a 2 + c y c l e , c o r r e s p o n d w i t h the syn thes i s o f p a r v a l b u m i n ( C e l i o a n d H e i z m a n n , 1982; B e r c h t o l d a n d M e a n s , 1985) . A n a l y s i s o f n a t i v e m y o s i n i s o e n z y m e a n d m y o s i n l i g h t c h a i n d i s t r i b u t i o n i n f a s t - t w i t c h m u r i n e d y s t r o p h i c m u s c l e ( F i t z s i m o n s a n d H o h , 1983; J a s c h a n d M o a s e , 1985) has s h o w n a n a l t e ra t i on i n these c o m p o n e n t s w h e n c o m p a r e d to t h e i r n o r m a l c o u n t e r p a r t s . T h e d y s t r o p h i c m u s c l e is c h a r a c t e r i z e d b y a n i nc rease i n s l o w a n d i n t e r m e d i a t e m y o s i n , a n d a dec rease i n the p r o p o r t i o n o f m y o s i n l i g h t c h a i n 158 3 ( fast ) a n d m y o s i n l i g h t - c h a i n 2 ( f a s t - p h o s p h o r y l a t e d ) . S a m a h a a n d T h i e s (1979) i n v e s t i g a t e d the m y o s i n l i g h t c h a i n s i n d y s t r o p h i c h u m a n m u s c l e a n d f o u n d no a l t e ra t i ons i n the t y p e o r m o b i l i t y o f L C I a n d L C 2 w h e n c o m p a r e d to n o r m a l m u s c l e . H o w e v e r , a m o r e d i v e r s e c o m p o s i t i o n o f m y o s i n l i g h t c h a i n s has b e e n d e t e c t e d i n t ype 1 f i b e r s o f D M D pa t ien ts t h a n i n c o n t r o l m u s c l e ( T a k a g i et a l . , 1982) . I n p a r t i c u l a r , the f a s t - t y p e L C 3 was n o t e d i n 5 5 % o f these s l o w t y p e f i b e r s . I n a s t u d y o f h u m a n s k e l e t a l i s o m y o s i n s , F i t z s i m o n s a n d H o h (1981) f o u n d la rge a m o u n t s o f f e t a l m y o s i n i n m u s c l e f r o m D u c h e n n e pa t ien ts \ a n d sugges ted that the p r e s e n c e o f th i s i s o f o r m re f l ec t s m u s c l e r e g e n e r a t i o n o r i m m a t u r i t y . T h e pe rcen tage o f f e t a l m y o s i n i n m u s c l e a f f e c t e d w i t h D u c h e n n e d y s t r o p h y was e s t i m a t e d b y these w o r k e r s at 1 - 1 2 % o f the to ta l a m o u n t o f m y o s i n . A m o r e recen t i m m u n o f l u o r e s c e n c e s t u d y ( S c h i a f f i n o et a l . , 1986) a lso r e p o r t e d the e x i s t e n c e o f m y o s i n i s o f o r m s that w e r e i n d i s t i n g u i s h a b l e f r o m f e t a l m y o s i n i n h u m a n d y s t r o p h i c m u s c l e . T h e s e w o r k e r s o b s e r v e d m a n y s m a l l e x t r a f u s a l f i b e r s that s t a i n e d p o s i t i v e l y f o r a n t i - f e t a l m y o s i n , a n d e s t i m a t e d the p e r c e n t a g e o f r e a c t i v e f i b e r s to be b e t w e e n 2 0 % a n d 4 1 % . T h e y sugges ted that f e t a l m y o s i n i n d y s t r o p h i c m u s c l e a re c o n f i n e d to a p o p u l a t i o n o f n e w l y f o r m e d f i b e r s r e s u l t i n g f r o m success i ve c y c l e s o f nec ros i s a n d r e g e n e r a t i o n . A l t e r n a t i v e l y , m u s c l e f i b e r m a t u r a t i o n that is n o r m a l l y a c c o m p a n i e d b y the cessa t i on o f f e t a l m y o s i n syn thes i s m a y be d e l a y e d i n the d y s t r o p h i c p rocess . P a r r y a n d D e s y p r i s (1983) a lso d e m o n s t r a t e d w i t h i m m u n o h i s t o c h e m i c a l m e t h o d s the p resence o f s l o w m y o s i n i n f a s t - t w i t c h h i n d l i m b m u s c l e s o f d y s t r o p h i c m i c e . I n a d d i t i o n to these f i n d i n g s , s i m i l a r i t i e s i n e n z y m e a n d i s o e n z y m e pa t te rns o f a n a e r o b i c m e t a b o l i s m i n b o t h a d u l t d y s t r o p h i c m o u s e m u s c l e a n d i m m a t u r e n o r m a l m u s c l e h a v e r e c e n t l y b e e n d e m o n s t r a t e d (Pe te l l et a l . , 1984; R e i c h m a n n a n d P e t t e , 1984) . A n i n c r e a s e d syn thes i s a n d a c c e l e r a t e d d e g r a d a t i o n o f m a n y o t h e r m u s c l e p r o t e i n s i n d y s t r o p h i c m u s c l e ( G a r b e r et a l . , 1980) m a y a c c o u n t f o r the p r e s e n c e o f i m m a t u r e m u s c l e c h a r a c t e r i s t i c s . T h e s e a b n o r m a l i t i e s co r re l a te w e l l w i t h the c o n t r a c t i l e a n d h i s t o c h e m i c a l c h a n g e s j us t m e n t i o n e d , i n d i c a t i n g a 159 t r a n s f o r m a t i o n i n e x t r a f u s a l f i b e r s to a m o r e i m m a t u r e o r i n t e r m e d i a t e state. F o r a m o r e c o m p l e t e s t u d y o f th is m a t u r a t i o n i s sue , i t w o u l d h a v e b e e n u s e f u l to i n c l u d e i n th is s t u d y t issue samp les f r o m 1 - w e e k - o l d d y s t r o p h i c m i c e a n d those f r o m a n o t h e r d e v e l o p m e n t a l s tage, s u c h as 8 - 1 2 w e e k s o f age . T h e s e age g roups w o u l d h a v e p r o v i d e d d a t a o n a m o r e i m m a t u r e stage o f d e v e l o p m e n t a n d m a y have r e v e a l e d t r a n s f o r m a t i o n s i n P V d i s t r i b u t i o n d u r i n g a t r a n s i t i o n a l s tage o f 8 - 1 2 w e e k s . D u e to the d i f f i c u l t i e s i n o b t a i n i n g adequa te n u m b e r s o f y o u n g d y s t r o p h i c a n i m a l s , a n d i n the t i m e c o n s t r a i n t s , s u c h a n e x t e n s i v e s t u d y was no t u n d e r t a k e n . W h y is t he re a r e d u c t i o n i n P V i n the d y s t r o p h i c f a s t - t w i t c h E D L m u s c l e c o n c o m i t a n t w i t h a n i n c r e a s e i n th is c a l c i u m - b i n d i n g p r o t e i n i n the s l o w - t w i t c h so leus , a n d w h a t are the f u n c t i o n a l i m p l i c a t i o n s o f these a l t e ra t i ons? A dec rease i n the p a r v a l b u m i n c o n t e n t is no t m e r e l y re l a ted to a d e p l e t i o n o f the p r o t e i n b u t to a c o n v e r s i o n i n f i b e r t ypes to a p o p u l a t i o n o f m u s c l e f i b e r s that c o n t a i n less p a r v a l b u m i n . T h e s e m y o f i b e r s a re no t n e c e s s a r i l y t y p e 1 / S O o r t y p e 2 / F O G f i b e r s bu t p e r h a p s e i t h e r a t y p i c a l , r e g e n e r a t i n g o r d e g e n e r a t i n g f i b e r s tha t d i s p l a y l o w p a r v a l b u m i n i m m u n o r e a c t i v i t y . P a r v a l b u m i n l o c a l i z a t i o n a n d i ts c o n t e n t i n f a s t - t w i t c h ske le ta l m u s c l e s r e s p o n d r a p i d l y to e x p e r i m e n t a l l y i n d u c e d f i b e r t y p e t r a n s f o r m a t i o n s ( K l u g et a l . , 1983a ; G r e e n et a l . , 1984; S t u h l f a u t h et a l . , 1984; M u n t e n e r et a l . , 1985). It has b e e n e s t a b l i s h e d as one o f the ear l ies t a n d mos t sens i t i ve m a r k e r s o f a c t i v i t y - i n d u c e d f a s t - t o - s l o w c o n v e r s i o n s i n d e v e l o p i n g ske le ta l m u s c l e ( K l u g et a l . , 1983a ,b ) . B e c a u s e a n a l t e r e d p a r v a l b u m i n con ten t has a lso b e e n o b s e r v e d i n response to e l e c t r i c a l s t i m u l a t i o n , c r o s s - r e i n n e r v a t i o n a n d d e n e r v a t i o n , i t is m o r e l i k e l y that the changes o b s e r v e d i n the d y s t r o p h i c m u r i n e m u s c l e a re s e c o n d a r y e f f e c t s a n d a c o n s e q u e n c e o f a c o n v e r s i o n i n the p h e n o t y p i c e x p r e s s i o n o f e x t r a f u s a l m y o f i b e r t ypes . We c a n n o t s p e a k o f m u s c l e f i b e r t r ans i t i ons based o n one p a r a m e t e r a l o n e , s u c h as m y o s i n A T P a s e f i b e r t y p e o r P V con ten t . T h e t r a n s f o r m a t i o n m u s t be c o n s i d e r e d at the l e v e l o f s p e c i f i c s y s t e m s , no t m e r e l y as a s i ng l e e n t i t y w i t h respec t to the 160 m u s c l e f i b e r . It has b e e n o b s e r v e d that f i b e r t y p e t r ans i t i ons i n v o l v e changes i n e n z y m e p r o f i l e s , s a r c o p l a s m i c r e t i c u l u m , m y o s i n i s o f o r m s , a n d P V ( G r e e n et a l . , 1984). T h e s e a l t e ra t i ons f o l l o w a n o r d e r e d t i m e c o u r s e as t h e y are d e v e l o p m e n t a l l y r e g u l a t e d . T h u s , i t w o u l d a p p e a r that m u r i n e d y s t r o p h y i n d u c e s , i n a g r a d e d f a s h i o n , the o b s e r v e d m o d i f i c a t i o n s o f the v a r i o u s f u n c t i o n a l sys tems o f the e x t r a f u s a l m u s c l e f i b e r s i m i l a r to the p l a s t i c i t y d e s c r i b e d i n response to o t h e r n e u r o m u s c u l a r c o n d i t i o n s (Pe t t e , 1980) . T h e c h a n g e s i n f i b e r d i s t r i b u t i o n i n the d y s t r o p h i c m u s c l e s o b s e r v e d i n the p resen t s t u d y , a n d i n p r e v i o u s r e p o r t s , m a y i n f a c t p l a y a c r u c i a l r o l e i n the d e c r e a s e d c a l c i u m u p t a k e o f the S R , i n a d d i t i o n to the a l t e ra t i ons i n P V . T y p e 2 fast f i b e r s h a v e a m o r e h i g h l y d e v e l o p e d S R t h a n t y p e 1 s l o w f i b e r s . A s d y s t r o p h i c m u s c l e a c q u i r e s a m o r e i n t e r m e d i a t e f i b e r t y p e d i s t r i b u t i o n , i t is p o s s i b l e the c a l c i u m u p t a k e a b i l i t y o f th is t issue m i r r o r s the p h e n o t y p i c e x p r e s s i o n i n f i b e r t ypes . A n i n c r e a s e i n the r e l a t i v e a m o u n t s o f i n t e r m e d i a t e o r s l o w m u s c l e f i b e r s , a d i p o s e t i ssue , a n d c o n n e c t i v e t issue w o u l d n a t u r a l l y a f f e c t the m u s c l e ' s o v e r a l l c a l c i u m m e t a b o l i s m . T h i s i n t u r n w o u l d be r e f l e c t e d i n the p r o l o n g e d h a l f - r e l a x a t i o n t imes a n d t i m e - t o - p e a k t e n s i o n that a re c h a r a c t e r i s t i c f ea tu res o f the d y s t r o p h i c p rocess i n the d y 2 J / d y 2 J s t r a i n o f m o u s e (B ress le r et a l . , 1983; P a r r y a n d D e s y p r i s , 1983) . T h e d e l a y e d p h y s i o l o g i c a l r eac t i ons h a v e been a t t r i b u t e d to the p r e s e n c e o f s l o w m y o s i n i n f a s t - t w i t c h m u s c l e ( P a r r y a n d D e s y p r i s , 1983) . H o w e v e r , these c h a n g e s a lso co r re la te w e l l w i t h the decreases i n P V con ten t . T h u s d y s t r o p h y appea rs to i n d u c e n u m e r o u s a s y n c h r o n o u s c h a n g e s i n the d i f f e r e n t i a t i o n p rocess as a c o n s e q u e n c e o f an u n k n o w n g e n e t i c d e f e c t . A l t h o u g h the c h a n g e s i n P V c o n t e n t i n the d y s t r o p h i c fas t m u s c l e m i r r o r s the p r o l o n g e d h a l f - r e l a x a t i o n t i m e o f th is m u s c l e , a n i n c r e a s e d P V c o n t e n t i n the s l o w m u s c l e is no t p a r a l l e l e d b y a dec rease i n i ts h a l f - r e l a x a t i o n t i m e b y 32 w e e k s o f age ( B r e s s l e r et a l . , 1983) . A s i m i l a r f i n d i n g was o b s e r v e d i n a recen t c r o s s - r e i n n e r v a t i o n s t u d y o f fas t a n d s l o w m u s c l e o f the rat ( M i i n t e n e r et a l . , 1986) . 161 C r o s s - r e i n n e r v a t e d fas t m u s c l e w i t h a s l o w n e r v e h a d s i m i l a r h i s t o c h e m i c a l a n d p h y s i o l o g i c a l c h a r a c t e r i s t i c s to those o f the n o r m a l s l o w m u s c l e . H o w e v e r , the P V c o n t e n t was 3 0 - 8 0 t imes g rea te r t h a n the s l o w m u s c l e . T h u s a s l o w c o n t r a c t i n g / s l o w r e l a x i n g m u s c l e m a y c o n t a i n m o r e P V t h a n a fas t c o n t r a c t i n g / f a s t r e l a x i n g m u s c l e i n the f a c e o f a d i s t u r b a n c e to i ts n o r m a l n e r v e - m u s c l e i n t e r a c t i o n ( M i i n t e n e r et a l . , 1986) . T h i s m i g h t suggest that P V is a u s e f u l m a r k e r f o r e a r l y changes i n n e u r o m u s c u l a r d i s t u r b a n c e s as w e l l as a n i n d i c a t o r o f a b n o r m a l c h a n g e s i n m u s c l e t r a n s f o r m a t i o n s . T h e s e o b s e r v a t i o n s a lso s u p p o r t the h y p o t h e s i s tha t m u r i n e d y s t r o p h y m a y s u f f e r f r o m an a l t e red n e r v e - m u s c l e i n t e r a c t i o n . A t the p resen t t i m e i t is no t c l ea r w h e t h e r the d i f f e r e n t m u s c l e p r o t e i n s are d y s f u n c t i o n a l i n a n y w a y i n the d y s t r o p h i c t i ssue. It is p o s s i b l e that the changes seen i n P V l o c a l i z a t i o n a n d con ten t i n the p resen t s t u d y m a y be the resu l t o f an a l t e r a t i o n i n the a n t i g e n i c i t y o f the p r o t e i n d u e to c h e m i c a l o r s t r u c t u r a l c h a n g e s . In l i g h t o f t h i s , i t m i g h t be u s e f u l to r u n a n a m i n o a c i d ana l ys i s a n d H P L C p r o f i l e o n P V e x t r a c t e d f r o m t y p e d s i ng l e f i b e r s o f d y s t r o p h i c m u s c l e . It is d i f f i c u l t to d e t e r m i n e w h a t e f f e c t a n a l t e r a t i o n i n the p a r v a l b u m i n c o n c e n t r a t i o n m a y h a v e o n the i n t r a c e l l u l a r C a 2 + - b u f f e r i n g s y s t e m o f s k e l e t a l m u s c l e . C e r t a i n l y a d i m i n i s h e d p r o t e i n con ten t w o u l d a l te r the C a 2 + - b u f f e r i n g c a p a c i t y o f the m u s c l e f i b e r , a n d p e r h a p s l e a d to an a c t i v a t i o n o f C a 2 + - d e p e n d e n t p ro teases , w h i c h i n t u r n c a n l e a d to the i r r e v e r s i b l e c e l l d e s t r u c t i o n t y p i c a l o f th is d i sease . K l u g a n d c o w o r k e r s (1985) h a v e e s t i m a t e d a 360 p m o l e / k g o f m u s c l e r e d u c t i o n i n the s a r c o p l a s m i c C a 2 + - b i n d i n g c a p a c i t y i n d y s t r o p h i c t i ssue. H o w e v e r , i t is no t k n o w n w h e t h e r the l eve l s o b s e r v e d i n d y s t r o p h i c f a s t - t w i t c h m u s c l e a re s i g n i f i c a n t l y r e d u c e d to be r e s p o n s i b l e f o r the i n c r e a s e d i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n s rioted i n these m u s c l e s . I n the d y s t r o p h i c s ta te , i t n o w seems p l a u s i b l e that the s a r c o p l a s m i c f r e e c a l c i u m c o n c e n t r a t i o n r ises i n response to: (1) a n a l t e red m e m b r a n e p e r m e a b i l i t y , (2) a n i m p a i r e d f u n c t i o n i n g o f the S R C a 2 + - u p t a k e a n d s e q u e s t e r i n g s y s t e m , a n d (3) a dec rease i n P V . 162 A dec rease i n the n u m b e r o f P V - p o s i t i v e e x t r a f u s a l f i b e r s , c o n c o m i t a n t w i t h a p r o l o n g e d h a l f - r e l a x a t i o n t i m e , h a v e a lso b e e n o b s e r v e d i n s e v e r a l n e u r o m u s c u l a r d i s t u r b a n c e s . S t u d i e s o n the m u t a n t m o u s e "a r res ted d e v e l o p m e n t o f r i g h t i n g response " as w e l l as those o n c r o s s - r e i n n e r v a t i o n a n d d e n e r v a t i o n o f ra t m u s c l e s , i n d i c a t e that the e x p r e s s i o n o f P V i n fas t s ke le ta l m u s c l e is d e p e n d e n t o n a n e r v e - m u s c l e i n t e r a c t i o n ( S t u h l f a u t h et a l . , 1984; M i i n t e n e r et a l . , 1985 , 1986) . It has been sugges ted that P V is u n d e r n e u r a l r e g u l a t i o n b y f a s t - t y p e m o t o r - n e u r o n a c t i v i t y s i nce c o n c e n t r a t i o n s o f P V a re s u p p r e s s e d i n p r e s u m p t i v e f a s t - t w i t c h m u s c l e b y d e n e r v a t i o n , w h e r e a s s l o w - t w i t c h m u s c l e appears u n a f f e c t e d b y d e n e r v a t i o n ( L e b e r e r a n d P e t t e , 1986b) . D e n e r v a t i o n o f d e v e l o p i n g f a s t - t w i t c h m u s c l e resu l ts i n a r a p i d d e c a y i n P V c o m p a r e d to that o f a d u l t fas t m u s c l e w h i c h e x h i b i t s a d e l a y e d s u p p r e s s i o n i n P V c o n c e n t r a t i o n s . T h i s d i f f e r e n c e i n the rate o f P V r e d u c t i o n was a t t r i b u t e d to the h i g h e r t u r n o v e r o f p r o t e i n s i n the d e v e l o p i n g m u s c l e ( L e b e r e r a n d P e t t e , 1986a) . T h e s e f i n d i n g s suggest that the changes o b s e r v e d i n d y s t r o p h i c m u s c l e m a y have i m p o r t a n t i m p l i c a t i o n s . T h e a l te ra t ions i n P V m a y r e f l e c t a loss o f the a c q u i r e d state o f d i f f e r e n t i a t i o n o f the a f f e c t e d m u s c l e s , e v i d e n c e d b y a t r a n s f o r m a t i o n i n f i b e r t ypes . T h i s i n t u r n m a y be d u e to a f a i l u r e o f the n o r m a l n e r v e - m u s c l e i n t e r a c t i o n i n pos tna ta l d e v e l o p m e n t as sugges ted b y J a s c h a n d M o a s e (1985) . In a s t u d y o f the t r o p h i c i n f l u e n c e s o f n e r v e a n d m u s c l e i n the d y 2 J m o u s e Sa i to a n d c o w o r k e r s (1983) f o u n d that the d y s t r o p h i c m u s c l e f i b e r s u n d e r w e n t d e g e n e r a t i o n i n s p i t e o f c o m p l e t e r e i n n e r v a t i o n b y a n o r m a l n e r v e , a n d n o r m a l m u s c l e c o u l d not p r e v e n t d e g e n e r a t i o n o f the d y s t r o p h i c n e r v e . N o m y o t r o p h i c i n f l u e n c e s w e r e o b s e r v e d . It a p p e a r e d that b o t h n e r v e a n d m u s c l e w e r e g e n e t i c a l l y p r o g r a m m e d to degene ra te i n m u r i n e d y s t r o p h y a n d b o t h c o u l d d o so to s o m e ex ten t i n d e p e n d e n t o f e a c h o the r . M u s c l e d e g e n e r a t i o n was the a d d i t i v e e f f e c t s o f the d y s t r o p h i c gene e x p r e s s i o n a n d a b n o r m a l n e u r o t r o p h i c i n f l u e n c e s . N e r v e d e g e n e r a t i o n , o n the o the r h a n d , was i n f l u e n c e d b y d y s t r o p h i c gene e x p r e s s i o n a l o n e . I n l i g h t o f t h i s , a b n o r m a l n e u r o t r o p h i c i n f l u e n c e s m a y , i n pa r t , be r e s p o n s i b l e f o r the a l t e red P V e x p r e s s i o n i n 163 d y s t r o p h i c t i s sue , as w e l l as the changes i n the S R a n d f i b e r c o m p o s i t i o n w h i c h ar ise as s e c o n d a r y f ea tu res g o v e r n e d b y the u n k n o w n p r i m a r y d e f e c t . A l t h o u g h the p h y s i o l o g i c a l ro le o f P V i n n e r v o u s t issue is no t k n o w n , P V i n th is t i ssue is b e l i e v e d to f u n c t i o n i n the C a 2 + - r e g u l a t e d re lease o f n e u r o t r a n s m i t t e r s a n d i n a x o n a l f l o w . T h e a u t h o r o f a recen t r epo r t ( C e l i o , 1986) has sugges ted that P V m a y f u n c t i o n to s h o r t e n the r e f r a c t o r y p e r i o d a n d e n a b l e G A B A n e u r o n s to f i r e i n r a p i d s u c c e s s i o n . It was a lso p r o p o s e d that the re lease o f M g 2 + b y P V m a y t r i gge r M g - d e p e n d e n t e n z y m e s w i t h i n the G A B A n e u r o n s , a n d t h e r e b y regu la te b o t h e l e c t r i c a l a n d m e t a b o l i c a c t i v i t y w i t h i n n e r v o u s t i ssue. In l i g h t o f the i n v o l v e m e n t o f P V i n n e u r o n a l p rocesses a n d i n ske le ta l m u s c l e , p a r v a l b u m i n s are p r o b a b l y i n v o l v e d i n the r e g u l a t i o n o f v a r i o u s C a 2 + - d e p e n d e n t sys tems . E v e n t h o u g h the d i s t r i b u t i o n a n d c o n c e n t r a t i o n o f P V is a l t e red i n b o t h the fas t a n d s l o w m u s c l e s o f the m u s c u l a r d y s t r o p h i c m i c e , i t is u n l i k e l y that the f u n c t i o n o r c o n f o r m a t i o n o f the p r o t e i n is m o d i f i e d . It w o u l d be o f in te res t to s t u d y the response o f P V i n the n e r v o u s t issue o f the d y s t r o p h i c a n i m a l s to d e t e r m i n e w h e t h e r the o b s e r v e d c h a n g e s rep resen t a m o r e g l o b a l a l t e r a t i o n i n P V o r i f t hey are s p e c i f i c to ske le ta l m u s c l e . I f p a r v a l b u m i n is r e g u l a t e d b y a n e u r o g e n i c f a c t o r , w h i c h i t appea rs to b e , t h e n changes i n i ts c o n t e n t o r d i s t r i b u t i o n m i g h t a lso be e x p e c t e d i n n e r v o u s t issue. A g l o b a l e f f e c t o n P V c o n c e n t r a t i o n w o u l d a d d c r e d e n c e to a d i s t u r b a n c e i n c a l c i u m r e g u l a t i o n as a m a j o r d e f e c t i n d y s t r o p h i c s k e l e t a l m u s c l e . 164 B I B L I O G R A P H Y A s h l e y , C C . 1983 C a l c i u m i n m u s c l e . In : C a l c i u m i n B i o l o g y . T . G . S p i r o , e d . J o h n W i l e y , N e w Y o r k , p p . 1 0 7 - 1 7 5 . A s h l e y , C C , G r i f f i t h s , P . J . 1983 T h e e f f e c t o f i n j e c t i o n o f p a r v a l b u m i n s i n t o s i ng le m u s c l e f i b e r s f r o m the b a r n a c l e B a l a n u s h i b i l u s . J . P h y s i o l . ( L o n d ) , 3 4 5 : 1 0 5 P . A t w o o d , H . L . , K w a n , I. 1978 D y s t r o p h i c a n d n o r m a l m i c e s h o w a g e - d e p e n d e n t d i v e r g - enc e o f m u s c l e s o d i u m c o n c e n t r a t i o n s . E x p . N e u r o l . , 6 0 : 3 8 6 - 3 9 2 . B a k k e r , G . J . , R i c h m o n d , F . J . R . 1981 T w o types o f m u s c l e s p i n d l e s i n ca t n e c k musc les : a h i s t o c h e m i c a l s t u d y o f i n t r a f u s a l f i b e r c o m p o s i t i o n . J . N e u r o p h y s i o l . , 4 5 : 9 7 3 - 9 8 6 . B a n d m a n , E . 1985 C o n t i n u e d e x p r e s s i o n o f n e o n a t a l m y o s i n h e a v y c h a i n i n a d u l t d y s t r o p h i c s k e l e t a l m u s c l e . S c i e n c e , 2 2 7 : 7 8 0 - 7 8 2 . B a n k e r , B . Q . , H i r s t , N . S . , C h e s t e r , C . S . , F o k , R . Y . 1979 H i s t o m e t r i c a n d e l e c t r o n c y t o c h e m i c a l s t u d y o f m u s c l e i n the d y s t r o p h i c m o u s e . A n n . N . Y . A c a d . S c i . , 3 1 7 : 1 1 5 - 1 3 2 . B a n k s , R . W . , H a r k e r , D . W . , S t a c e y , M . J . 1977 A s t u d y o f m a m m a l i a n i n t r a f u s a l m u s c l e f i b e r s u s i n g a c o m b i n e d h i s t o c h e m i c a l a n d u l t r a s t r u c t u r a l t e c h n i q u e . J . A n a t . , 1 2 3 : 7 8 3 - 7 9 6 . B a r k e r , D . 1974 T h e m o r p h o l o g y o f m u s c l e r ecep to r s . In : H a n d b o o k o f S e n s o r y P h y s i o l o g y . V o l . 1 1 1 / 2 , M u s c l e R e c e p t o r s . C C H u n t , e d . S p r i n g e r - V e r l a g , B e r l i n , p p . 8 4 - 8 9 . B a r k e r , D . , C h i n , N . K . 1960 T h e n u m b e r a n d d i s t r i b u t i o n o f m u s c l e s p i n d l e s i n c e r t a i n m u s c l e s o f the ca t . J . A n a t . , 9 4 : 4 7 3 - 4 8 6 . B a r k e r , D . , M i l b u r n , A . 1984 D e v e l o p m e n t a n d r e g e n e r a t i o n o f m a m m a l i a n m u s c l e s p i n d l e s . S c i . P r o g . , 6 9 : 4 5 - 6 4 . B a r n a r d , E . A . , B h a r g a v a , A . K . , H u d e c k i , M . S . 1976 P o s t p o n e m e n t o f s y m p t o m s o f h e r e d i t a r y m u s c u l a r d y s t r o p h y i n c h i c k e n s b y 5 - h y d r o x y t r y p t a m i n e an tagon is t s . N a t u r e , 263 : 4 2 2 - 4 2 4 . B a r o n , G . , D e m a i l l e , J . D u t r u g e , E . 1975 T h e d i s t r i b u t i o n o f p a r v a l b u m i n i n m u s c l e a n d o the r t i ssues. F E B S L e t t . , 5 6 : 1 5 6 - 1 6 0 . B a y l o r , S . M . , C h a n d l e r , W . K . , M a r s h a l l , M . W . 1983 S a r c o p l a s m i c r e t i c u l u m c a l c i u m re lease i n f r o g s k e l e t a l m u s c l e f i b e r s e s t i m a t e d f r o m A r s e n a z o 111 c a l c i u m t rans ien ts . J . P h y s i o l . ( L o n d ) , 3 4 4 : 6 2 5 - 6 6 6 . B e n z o n a n a , G . , C a p o n y , J . P . , P e c h e r e , J . F . 1972 T h e b i n d i n g o f c a l c i u m to m u s c u l a r p a r v a l b u m i n s . B i o c h i m . B i o p h y s . A c t a , 2 7 8 : 1 1 0 - 1 1 6 . B e n z o n a n a , G . , C h a p o n n i e r , C , G a b b i a n i , G . 1977 I m m u n o f l u o r e s c e n t s u b c e l l u l a r l o c a l i z a t i o n o f m u s c l e p o r t e i n s . E x p e r i e n t i a , 33 :787 . 165 B e n z o n a n a , G . , W n u k , W . , C o x , J . A . , G a b b i a n i , G . 1975 C e l l u l a r d i s t r i b u t i o n o f s a r c o p l a s m i c c a l c i u m - b i n d i n g p r o t e i n s b y i m m u n o f l u o r e s c e n c e . H i s t o c h e m i s t r y , 5 1 : 3 3 5 - 3 4 1 . B e r c h t o l d , M . W . , C e l i o , M . R . , H e i z m a n n , C . W . 1984 P a r v a l b u m i n i n n o n - m u s c l e t issues o f the rat: q u a n t i t a t i o n a n d i m m u n o h i s t o c h e m i c a l l o c a l i z a t i o n . J . B i o l . C h e m . , 2 5 9 : 5 1 8 9 - 5 1 9 6 . B e r c h t o l d , M . W . , C e l i o , M . R . , H e i z m a n n , C . W . 1985 P a r v a l b u m i n i n h u m a n b r a i n . J . N e u r o c h e m . , 4 5 : 2 3 5 - 2 3 9 . B e r c h t o l d , M . W . , H e i z m a n n , C . W . , W i l s o n , K . J . 1982a P r i m a r y s t r u c t u r e o f p a r v a l b u m i n f r o m rat s k e l e t a l m u s c l e . E u r . J . B i o c h e m . , 1 2 7 : 3 8 1 - 3 8 9 . B e r c h t o l d , M . W . , H e i z m a n n , C . W . , W i l s o n , K . J . 1983 C a 2 + - b i n d i n g p ro te i ns : A c o m p a r a t i v e s t u d y o f t h e i r b e h a v i o r d u r i n g h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y u s i n g g r a d i e n t e l u t i o n o n r e v e r s e - p h a s e s u p p o r t s . A n a l . B i o c h e m . , 1 2 9 : 1 2 0 - 1 3 1 . B e r c h t o l d , M . W . , M e a n s , A . R . 1985 T h e C a 2 + - b i n d i n g p r o t e i n p a r v a l b u m i n : m o l e c u l a r c l o n i n g a n d d e v e l o p m e n t a l r e g u l a t i o n o f m R N A a b u n d a n c e . P r o c . N a t l . A c a d . S c i . U S A , 8 2 : 1 4 1 4 - 1 4 1 8 . B e r c h t o l d , M . W . , W i l s o n , K . J . , H e i z m a n n , C . W . 1982b I so la t i on o f n e u r o n a l p a r v a l b u m i n b y h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y . C h a r a c t e r i z a t i o n a n d c o m p a r i s o n w i t h m u s c l e p a r v a l b u m i n . B i o c h e m . , 2 1 : 6 5 5 2 - 6 5 5 7 . B l u m , H . E . , L e h k y , P . , K o h l e r , L . , S t e i n , E . A . , F i s c h e r , E . H . 1977 C o m p a r a t i v e p r o p e r t i e s o f ve r t eb ra te p a r v a l b u m i n . J . B i o l . C h e m . , 2 5 2 : 2 8 3 4 - 2 8 3 8 . B o y d , I .A. , S m i t h , R . S . 1984 T h e m u s c l e s p i n d l e . In : P e r i p h e r a l N e u r o p a t h y , V o l . 1. P . J . D y c k , P . K . T h o m a s , E . H . L a m b e r t , R . B u n g e , eds . W . B . S a u n d e r s , P h i l a d e l p h i a , p p . 1 7 1 - 2 0 2 . B r a y , G . M . , P e r k i n s , S . , P e t e r s o n , A . C , A g u a y o , A . J . 1977 S c h w a n n c e l l m u l t i p l i c a t i o n d e f i c i t i n n e r v e roo ts o f n e w b o r n d y s t r o p h i c m i c e . J . N e u r o l . S c i . , 3 2 : 2 0 3 - 2 1 2 . B r e s s l e r , B . H . , J a s c h , L . G . , O v a l l e , W . K . , S l o n e c k e r , C . E . 1983 C h a n g e s i n i s o m e t r i c c o n t r a c t i l e p r o p e r t i e s o f f a s t - t w i t c h a n d s l o w - t w i t c h s k e l e t a l m u s c l e o f C 5 7 B L / 6 J d y 2 J / d y 2 J d y s t r o p h i c m i c e d u r i n g pos tna ta l d e v e l o p m e n t . E x p . N e u r o l . , 8 0 : 4 5 7 - 4 7 0 . B r i g g s , N . 1975 I d e n t i f i c a t i o n o f the s o l u b l e r e l a x i n g f a c t o r as a p a r v a l b u m i n . F e d . P r o c , 34 :540 . B r o o k e , M . H . , K a i s e r , K . K . 1970 M u s c l e f i b e r t ypes : H o w m a n y a n d w h a t k i n d ? A r c h . N e u r o l . , 2 3 : 3 6 9 - 3 7 9 . B u l f i e l d , G . , S i l l e r , W . G . , W i g h t , P . A . L . , M o o r e , K . J . 1984 X - c h r o m o s o m e - l i n k e d m u s c u l a r d y s t r o p h y ( m d x ) i n the m o u s e . P r o c . N a t l . A c a d . S c i . , 8 1 : 1 1 8 9 - 1 1 9 2 . B u t c h e r , L . A . , T o m k i n s , J . K . 1986 P r o t e i n p r o f i l e s o f s a r c o p l a s m i c r e t i c u l u m f r o m n o r m a l a n d d y s t r o p h i c m o u s e m u s c l e . J . N e u r o l . S c i . , 7 2 : 1 5 9 - 1 6 9 . 166 B u t l e r , J . , C o s m o s , E . 1977 H i s t o c h e m i c a l a n d s t r u c t u r a l a n a l y s i s o f the p h e n o t y p i c e x p r e s s i o n o f the d y s t r o p h i c gene i n the 1 2 9 / R e J d y / d y a n d the C 5 7 B L / 6 J d y 2 J / d y 2 J m i c e . E x p . N e u r o l . , 5 7 : 6 6 6 - 6 8 1 . B u t l e r , J . E . , F e l d b u s h , T . L . , M c G i v e r n , P . L . , S t e w a r t , N . 1978 T h e e n z y m e - l i n k e d i m m u n o s o r b e n t assay ( E L I S A ) : a m e a s u r e o f a n t i b o d y c o n c e n t r a t i o n o r a f f i n i t y . I m m u n o c h e m . , 1 5 : 1 3 1 - 1 3 6 . 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C h a r a c t e r i z a t i o n o f c o m p o n e n t I, i so l a t ed f r o m r e d m u s c l e s o f C v o r i n u s c a r p i o L . B i o c h i m . B i o p h y s . A c t a , 532: 2 9 4 - 3 0 4 . G r e e n , H . J . , K l u g , G . A . , R e i c h m a n n , H . , S e d o r f , U . , W i e h r e r , W . , P e t t e , D . 1984 E x e r c i s e - i n d u c e d f i b e r t y p e t r ans i t i ons w i t h r e g a r d to m y o s i n , p a r v a l b u m i n , a n d s a r c o p l a s m i c r e t i c u l u m i n m u s c l e o f the rat . P f l u g e r s A r c h . , 4 0 0 : 4 3 2 - 4 3 8 . G r e e n , H . J . , R e i c h m a n n , H . , P e t t e , D . 1982 A c o m p a r i s o n o f t w o A T P a s e b a s e d schemes f o r h i s t o c h e m i c a l m u s c l e f i b e r t y p i n g i n v a r i o u s m a m m a l s . H i s t o c h e m i s t r y , 7 6 : 2 1 - 3 1 . G r e e n w o o d , F . C . , H u n t e r , W . M . 1963 T h e p r e p a r a t i o n o f 1 3 1 1 - l a b e l l e d h u m a n g r o w t h h o r m o n e o f h i g h s p e c i f i c a c t i v i t y . B i o c h e m . J . , 8 9 : 1 1 4 - 1 2 3 . G u t h , L . , S a m a h a , F . J . 1970 P r o c e d u r e f o r the h i s t o c h e m i c a l d e m o n s t r a t i o n o f a c t o m y o s i n A T P a s e . E x p . N e u r o l . , 2 8 : 3 6 5 - 3 6 7 . H a i e c h , J , D e r a n c o u r t , J . , P e c h e r e , J . F . , D e m a i l l e , J . G . 1979a M a g n e s i u m a n d c a l c i u m b i n d i n g : E v i d e n c e f o r d i f f e r e n c e s b e t w e e n p a r v a l b u m i n s a n d a n e x p l a n a t i o n o f t he i r r e l a x i n g f u n c t i o n . B i o c h e m i s t r y , 18: 2 7 5 2 - 2 7 5 8 . H a i e c h , J . , D e r a n c o u r t , J . , P e c h e r e , J . F . , D e m a i l l e , J . G . 1979b A n e w l a r g e - s c a l e p u r i f i c a t i o n p r o c e d u r e f o r m u s c u l a r p a r v a l b u m i n s . B i o c h i m i e , 6 1 : 5 8 3 - 5 8 7 . H a m o i r , G . , G e r a r d i n - O t t h i e r s , N . , F o c a n t , B . , 1 9 8 0 P r o t e i n d i f f e r e n t i a t i o n o f the s u p e r f a s t s w i m b l a d d e r m u s c l e o f the t o a d f i s h Q p s a n u s tau . J . M o l . B i o l . , 1 4 3 : 1 5 5 - 1 6 0 . H a r r i s , J . B . , S la te r , C . R . 1980 A n i m a l m o d e l s : W h a t is t h e i r r e l e v a n c e to the pa thogenes i s o f h u m a n m u s c u l a r d y s t r o p h y ? B r . M e d . B u l l . , 3 6 : 1 9 3 - 1 9 7 . H e i l m a n n , C , P e t t e , D . 1979 M o l e c u l a r t r a n s f o r m a t i o n s i n s a r c o p l a s m i c r e t i c u l u m o f f a s t - t w i t c h m u s c l e b y e l e c t r o - s t i m u l a t i o n . E u r . J . B i o c h e m . , 9 3 : 4 3 7 - 4 4 6 . H e i z m a n n , C . W . 1984 P a r v a l b u m i n , a n i n t r a c e l l u l a r c a l c i u m - b i n d i n g p r o t e i n ; D i s t r i b u t i o n , p r o p e r t i e s a n d poss ib l e ro les i n m a m m a l i a n ce l l s . E x p e r i e n t i a , 4 0 : 9 1 0 - 9 2 1 . H e i z m a n n , C . W . , B e r c h t o l d , H . W . , R o w l e r s o n , A . M . 1982 C o r r e l a t i o n o f p a r v a l b u m i n c o n c e n t r a t i o n w i t h r e l a x a t i o n s p e e d i n m a m m a l i a n m u s c l e s . P r o c . N a t l . A c a d . S c i . U S A , 7 9 : 7 2 4 3 - 7 2 4 7 . H e i z m a n n , C . W . , C e l i o , M . R . 1986 L o c a l i z a t i o n o f the C a 2 + b i n d i n g p a r v a l b u m i n . M e t h o d s E n z y m o l . , i n press H e i z m a n n , C . W . , S t r e h l e r , E . E . 1979 C h i c k e n p a r v a l b u m i n - l i k e p r o t e i n a n d th ree o the r c o m p o n e n t s ( M r = 8 ,000 to 13,000) . J . B i o l . 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P . 1981 C a 2 + - e x c h a n g e w i t h t r o p o n i n a n d the r e g u l a t i o n o f m u s c l e c o n t r a c t i o n In : T h e R e g u l a t i o n o f M u s c l e C o n t r a c t i o n : E x c i t a t i o n - C o n t r a c t i o n C o u p l i n g . A . D . G r i n n e l l a n d M . A . B . B r a z i e r , eds . A c a d e m i c P r e s s , N e w Y o r k , p p . 2 4 1 - 2 5 9 . J o n e s , E . , K r o h n , P . 1960 O r t h o t o p i c o v a r i a n t r a n s p l a n t a t i o n i n m i c e . J . E n d o c r i n o l o g y , 2 0 : 1 3 5 - 1 4 6 . K a y , B . K . , S h a h , A . J . , H a l s t e a d , W . E . 1987 E x p r e s s i o n o f the c a l c i u m - b i n d i n g p r o t e i n , p a r v a l b u m i n , d u r i n g e m b r y o n i c d e v e l o p m e n t o f the f r o g X e n o p u s l a e v i s . J . C e l l B i o l . , 1 0 4 : 8 4 1 - 8 4 7 . K e l l y , B . S . , L e v y , J . G . , S i k o r a , L . 1979 T h e use o f the e n z y m e - l i n k e d i m m u n o s o r b e n t assay ( E L I S A ) f o r the d e t e c t i o n a n d q u a n t i f i c a t i o n o f s p e c i f i c a n t i b o d y f r o m c e l l c u l t u r e s . I m m u n o l o g y , 3 7 : 4 5 - 5 2 . K e n n e d y , W . R . , P o p p e l e , R . E . , Q u i c k , D . C . 1980 M a m m a l i a n m u s c l e s p i n d l e s . In : T h e P h y s i o l o g y o f P e r i p h e r a l N e r v e D i seases . A . J . S u m n e r , e d . W . B . S a u n d e r s C o . , P h i l a d e l p i a . p p . 7 4 - 1 3 3 . K h a n , M . A . , S o u k u p , T . 1979 A h i s t o e n z y m a t i c s t u d y o f ra t i n t r a f u s a l m u s c l e f i b e r s . H i s t o c h e m i s t r y , 6 2 : 1 7 9 - 1 8 9 . K l a m u t , H . J . , L i n , C . H . , S t r i c k l a n d , K . 1983 C a l m o d u l i n c o n t e n t a n d C a - a c t i v a t e d p ro tease a c t i v i t y i n d y s t r o p h i c h a m s t e r m u s c l e s . M u s c l e N e r v e , 6 :436 -441 . K l u g , G . , R e i c h m a n n , H . , P e t t e , D . 1983a R a p i d r e d u c t i o n i n p a r v a l b u m i n c o n c e n t r a t i o n d u r i n g c h r o n i c s t i m u a l t i o n o f r a b b i t f a s t - t w i t c h m u s c l e . F E B S L e t t e r s 1 5 2 : 1 8 0 - 1 8 2 . K l u g , G . , R e i c h m a n n , H . , P e t t e , R . 1985 D e c r e a s e d p a r v a l b u m i n con ten ts i n ske le ta l m u s c l e s o f C 5 7 B L / 6 J ( d y 2 J / d y 2 J ) d y s t r o p h i c m i c e . M u s c l e N e r v e , 8 : 5 7 6 - 5 7 9 . 171 K l u g , G . , W i e h r e r , W . , R e i c h m a n n , H . , L e b e r e r , E . , P e t t e , D . 1983b R e l a t i o n s h i p s b e t w e e n e a r l y a l t e ra t i ons i n p a r v a l b u m i n s , s a r c o p l a s m i c r e t i c u l u m a n d m e t a b o l i c e n z y m e s i n c h r o n i c a l l y s t i m u l a t e d f a s t - t w i t c h m u s c l e . P f l u g e r s A r c h . , 3 9 9 : 2 8 0 - 2 8 4 . K l u g h , H . E . 1974 S ta t i s t i cs : T h e essent ia ls f o r r e s e a r c h . J o h n W i l e y , N e w Y o r k , p p . 3 9 6 - 3 9 9 . K o z e k a , K . , O n t e l l , M . 1981 T h e t h r e e - d i m e n s i o n a l c y t o a r c h i t e c t u r e o f d e v e l o p i n g m u r i n e m u s c l e s p i n d l e s . D e v . B i o l . , 8 7 : 1 3 3 - 1 4 7 . et K r o n n i e , G . , D o n s e l a a r , Y . , S o u k u p , T . , v a n R a a m s d o n k , W . 1981 I m m u n o h i s t o c h e m i c a l d i f f e r e n c e s i n m y o s i n c o m p o s i t i o n a m o n g i n t r a f u s a l m u s c l e f i b e r s . H i s t o c h e m i s t r y , 7 3 : 6 5 - 7 4 . et K r o n n i e , G . , D o n s e l a a r , Y . , S o u k u p , T . , Z e l e n a , J . 1982 D e v e l o p m e n t o f i m m u n o h i s t o c h e m i c a l c h a r a c t e r i s t i c s o f i n t r a f u s a l f i b e r s i n n o r m a l a n d d e - e f f e r e n t e d rat m u s c l e s p i n d l e s . H i s t o c h e m i s t r y , 7 4 : 3 5 5 - 3 6 6 . K u c e r a , J . 1977 H i s t o c h e m i s t r y o f i n t r a f u s a l m u s c l e f i b e r s o u t s i d e the s p i n d l e c a p s u l e . A m . J . A n a t . , 1 4 8 : 4 2 7 - 4 3 2 . 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L e b e r e r , E . , P e t t e , D . 1986b N e u r a l r e g u l a t i o n o f p a r v a l b u m i n e x p r e s s i o n i n m a m m a l i a n ske le ta l m u s c l e . B i o c h e m . J . , 2 3 5 : 6 7 - 7 3 . L e h k y , P . , B l u m , H . E . , S t e i n , E . A . , F i s c h e r , E . H . 1974 I so la t i on a n d c h a r a c t e r i z a t i o n o f p a r v a l b u m i n s f r o m s k e l e t a l m u s c l e o f h i g h e r ve r teb ra tes . J . B i o l . C h e m . , 2 4 9 : 4 3 3 2 - 4 3 3 4 . L e P e u c h , C . J . , F e r r a z , C , W a l s h , M . P . , D e m a i l l e , J . G . , F i s c h e r , E . H . 1979 C a l c i u m a n d c y c l i c n u c l e o t i d e d e p e n d e n t r e g u l a t o r y m e c h a n i s m s d u r i n g d e v e l o p m e n t o f c h i c k e m b r y o s k e l e t a l m u s c l e . B i o c h e m . , 1 8 : 5 2 6 7 - 5 2 7 3 . 172 M a d r i d , R . E . , J a r o s , E . , C u l l e n , M . J . , B r a d l e y , W . G . 1 9 7 5 G e n e t i c a l l y d e t e r m i n e d d e f e c t o f S c h w a n n c e l l basemen t m e m b r a n e i n d y s t r o p h i c m o u s e . N a t u r e , 2 5 7 : 3 1 9 - 3 2 1 . M a r t o n o s i , A . N . 1982 R e g u l a t i o n o f c y t o p l a s m i c c a l c i u m c o n c e n t r a t i o n b y s a r c o p l a s m i c r e t i c u l u m . In : D i s o r d e r s o f the M o t o r U n i t . D . L . S c h o t l a n d , e d . J o h n W i l e y , N e w Y o r k , p p . 5 6 5 - 5 8 3 . M a y n a r d , J . A . , C o o p e r , R . R . , I o n a e s e u , V . V . 1977 A n u l t r a s t r u c t u r e i n v e s t i g a t i o n o f i n t r a f u s a l m u s c l e f i b e r s i n m y o t o n i c d y s t r o p h y . V i r c h o w s . A r c h . A . P a t h . A n a t . H i s t o l . , 3 7 3 : 1 - 1 3 . M e i e r , H . 1969 T h e m u s c l e s p i n d l e i n m i c e w i t h h e r e d i t a r y n e u r o m u s c u l a r d iseases. E x p e r i e n t i a , 2 5 : 9 6 5 - 9 6 8 . 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J . , K u r n i t , D . M . , K u n k e l , L . M . 1986 I so la t i on o f c a n d i d a t e c D N A s f o r p o r t i o n s o f the D u c h e n n e m u s c u l a r d y s t r o p h y gene . N a t u r e , 3 2 3 : 6 4 6 - 6 5 0 . M o n t g o m e r y , A . , S w e n a r c h u k , L . 1 9 7 8 F u r t h e r o b s e r v a t i o n s o n m y l i n a t e d a x o n n u m b e r i n n o r m a l a n d d y s t r o p h i c m i c e . J . N e u r o l . S c i . , 3 8 : 7 7 - 8 2 . M r a k , R . E . I 9 8 5 M u s c u l a r D y s t r o p h y In : M u s c l e M e m b r a n e s i n D i seases o f M u s c l e . R . E . M r a k e d . C R C Press I nc . , F l o r i d a , p p . 2 3 - 8 0 . M r a k , R . E . , B a s k i n , R . J . 1978 C a l c i u m t ranspo r t a n d p h o s p h o e n z y m e f o r m a t i o n i n d y s t r o p h i c m o u s e s a r c o p l a s m i c r e t i c u l u m . B i o c h e m . M e d . , 1 9 : 4 7 - 7 0 . M i i n t e n e r , M . , B e r c h t o l d , M . W . , H e i z m a n n , C . 1985 P a r v a l b u m i n i n c r o s s - r e i n n e r v a t e d a n d d e n e r v a t e d m u s c l e s . M u s c l e N e r v e , 8 : 1 3 2 - 1 3 7 . M i i n t e n e r , M . , R o w l e r s o n , A . M . , B e r c h t o l d , M . W . , H e i z m a n n , C . W . 1986 C h a n g e s i n the c o n c e n t r a t i o n o f the c a l c i u m - b i n d i n g p a r v a l b u m i n i n c r o s s - r e i n n e r v a t e d rat m u s c l e s . J . B i o l . C h e m . , 2 6 2 : 4 6 5 - 4 6 9 . N o c k o l d s , C . E . , K r e t s i n g e r , R . H . , C o f f e e , C . J . , B r a d s h a w , R . A . 1972 S t r u c t u r e o f a c a l c i u m - b i n d i n g c a r p m y o g e n . P r o c . N a t l . A c a d . S c i . U . S . A . , 6 9 : 5 8 1 - 5 8 4 . O ' F a r r e l l , P . H . 1975 H i g h r e s o l u t i o n t w o - d i m e n s i o n a l e l e c t r o p h o r e s i s o f p r o t e i n s . J . B i o l . 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O v a l l e , W . K . , B r e s s l e r , B . H . , J a s c h , L . G . , S l o n e c k e r , C . E . 1983 A b n o r m a l d i s t r i b u t i o n o f f i b e r t ypes i n the s l o w - t w i t c h so leus m u s c l e o f the C 5 7 B L / d y 2 J / d y 2 J d y s y r o p h i c m o u s e d u r i n g pos tna ta l d e v e l o p m e n t . A m . J . A n a t . , 1 6 8 : 2 9 1 - 3 0 4 . O v a l l e , W . K . , D o w , P . R . 1986 A l t e r a t i o n s i n m u s c l e s p i n d l e m o r p h o l o g y i n a d v a n c e d stages o f m u r i n e m u s c u l a r d y s t r o p h y . A n a t . R e c , 2 1 6 : 1 1 1 - 1 2 6 . O v a l l e , W . K . , S m i t h , R . S . 1972 H i s t o c h e m i c a l i d e n t i f i c a t i o n o f th ree t ypes o f i n t r a f u s a l m u s c l e f i b e r s i n the ca t a n d m o n k e y based o n the m y o s i n A T P a s e r e a c t i o . C a n . J . P h y s i o l . P h a r m . , 5 0 : 1 9 5 - 2 0 2 . P a r r y , D . J . , D e s y p r i s , G . 1983 S l o w i n g o f t w i t c h o f d y s t r o p h i c m o u s e m u s c l e is p a r t i a l l y d u e to a l t e red a c t i v i t y p a t t e r n . M u s c l e N e r v e , 6 : 3 9 7 - 4 0 7 . P a r r y , D . J . , P a r s l o w H . G . 1981 F i b e r t y p e s u s c e p t i b i l i t y i n d y s t r o p h i c m o u s e . E x p . N e u r o l . , 7 3 : 6 7 4 - 6 8 5 . P a r s l o w , H . G . , P a r r y , D .J .1981 S l o w i n g o f f a s t - t w i t c h m u s c l e i n the d y s t r o p h i c m o u s e . E x p . N e u r o l . , 7 3 : 6 8 6 - 6 9 9 . P e a r s e , A . G . E . 1968 H i s t o c h e m i s t r y . T h e o r e t i c a l a n d A p p l i e d . V o l . 1 . L i t t l e B r o w n & C o . , B o s t o n , p p . 5 9 - 6 9 . P e a r s e , A . G . E . 1980 H i s t o c h e m i s t r y , T h e o r e t i c a l a n d A p p l i e d , V o l . I. C h u r c h i l l L i v i n g s t o n e , N e w Y o r k , p p . 9 2 - 9 4 . P e c h e r e , J . F . 1968 M u s c u l a r p a r v a l b u m i n s as h o m o l o g o u s p r o t e i n s . C o m p . B i o c h e m . P h y s i o l . , 2 4 : 2 8 9 - 2 9 5 . P e c h e r e , J . F . 1974 I so lemen t d ' u n e p a r v a l b u m i n e d u m u s c l e de l a p i n . C R . A c a d . S c i . P a r i s , 2 7 8 : 2 5 7 7 - 2 5 7 9 . P e c h e r e , J . F . , C a p o n y , J . P . 1969 A c o m p a r i s o n at the p e p t i d e l e v e l o f m u s c u l a r p a r v a l b u m i n s f r o m s e v e r a l l o w e r ve r teb ra tes . C o m p . B i o c h e m . P h y s i o l . , 2 8 : 1 0 8 9 - 1102. P e c h e r e , J . F . , C a p o n y , J . P . , D e m a i l l e , J . 1973 E v o l u t i o n a r y aspec ts o f the s t r u c t u r e o f m u s c u l a r p a r v a l b u m i n s . S y s t e m Z o o l . , 2 2 : 5 3 3 - 5 4 8 . P e c h e r e , J . F . , D e m a i l l e , J . , C a p o n y , J . P . 1971 M u s c u l a r p a r v a l b u m i n s : P r e p a r a t i v e a n d a n a l y t i c a l m e t h o d s o f g e n e r a l a p p l i c a b i l i t y . B i o c h i m . B i o p h y s . A c t a , 2 3 6 : 3 9 1 - 4 0 8 . 174 P e c h e r e , J . F . , D e r a n c o u r t , J . , H a i e c h , J . 1977 T h e p a r t i c i p a t i o n o f p a r v a l b u m i n s i n the a c t i v a t i o n - r e l a x a t i o n c y c l e o f ve r teb ra te fas t s k e l e t a l m u s c l e . F E B S L e t t . , 7 5 : 1 1 1 - 1 1 4 . P e t e l l , J . K . , M a r s h a l l , N . A . , L e b h e r z , H . G . 1984 C o n t e n t a n d s y n t h e s i s o f seve ra l a b u n d a n t g l y c o l y t i c e n z y m e s i n ske le ta l m u s c l e s o f n o r m a l a n d d y s t r o p h i c m i c e . Int . J . B i o c h e m . , 1 6 : 6 1 - 6 7 . P e t e r , J . B . , B a r n a r d , R . J . , E d g e r t o n , V . R . , G i l l e s p i e , C . A . , S t e m p e l , K . E . 1972 M e t a b o l i c p r o f i l e s o f t h ree f i b e r t ypes o f s ke le ta l m u s c l e i n g u i n e a p i g s a n d rabb i t s . B i o c h e m . , 1 1 : 2 6 2 7 - 2 6 3 3 . P e t t e , D . 1980 P l a s t i c i t y o f M u s c l e . W a l t e r de G r u y t e r , B e r l i n . P e t t e , D . , K l u g , G . , R e i c h m a n n , H . 1985 P a r v a l b u m i n r e d u c t i o n i n r e l a t i o n to p o s s i b l e p e r t u r b a t i o n s o f C a 2 + - h o m e o s t a s i s i n m u s c u l a r d y s t r o p h y . A d v . E x p . M e d . B i o l . , 1 8 2 : 2 6 5 - 2 6 7 . P f y f f e r , G . E . , B o l o g a , E . , H e r s c h o w i t z , N . , H e i z m a n n , C . W . 1984 P a r v a l b u m i n , a n e u r o n a l p r o t e i n o f b r a i n c e l l c u l t u r e s . J . N e u r o c h e m . , 4 3 : 4 9 - 5 7 . P i e r o b o n B o r m i o l i , S . , S a r t o r e , S . , V i t a d e l l o , M . , S c h i a f f i n o , S. 1980 S l o w m y o s i n s i n ve r t eb ra te ske le ta l m u s c l e . A n i m m u n o f l u o r e s c e n c e s t u d y . J . C e l l B i o l . , 8 5 : 6 7 2 - 6 8 1 . P l a t z e r , A . 1979 E m b r y o l o g y o f t w o m u r i n e m u s c l e d iseases: M u s c u l a r d y s t r o p h y a n d m u s c u l a r d y s g e n e s i s . A n n . N . Y . A c a d . S c i . , 3 1 7 : 9 4 - 1 1 3 . P l a t z e r , A . , P o w e l l , J . A . 1975 F i n e s t r u c t u r e o f p r e n a t a l a n d e a r l y pos tna ta l d y s t r o p h i c m o u s e m u s c l e . J . N e u r o l . S c i . , 2 4 : 1 0 9 - 1 2 6 . P o t t e r , J . D . , J o h n s o n , D . , D e d m a n , J . R . , S c h r e i b e r , W . E . , M a n d e l , F . , J a c k s o n , R . L . , M e a n s , A . R . 1977 C a l c i u m - b i n d i n g p ro te ins : R e l a t i o n s h i p o f b i n d i n g , s t r u c t u r e , c o n f o r m a t i o n a n d b i o l o g i c a l f u n c t i o n . In : C a l c i u m - B i n d i n g P r o t e i n s a n d C a l c i u m F u n c t i o n . R . H . W a s s e r m a n et a l . , eds . E l s e v i e r N o r t h - H o l l a n d , N e w Y o r k , p p . 2 3 9 - 2 5 0 . R e i c h m a n n , H . , P e t t e , D . 1984 E n z y m e a c t i v i t i e s a n d a c t i v i t y p r o f i l e s i n m u s c l e f i b e r s o f d y s t r o p h i c , i m m a t u r e - n o r m a l , a n d a d u l t - n o r m a l B L 6 m i c e . M u s c l e N e r v e , 7 : 1 2 1 - 1 2 6 . R o b e r t s o n , S . P . , J o h n s o n , J . D . , P o t t e r , J . D . 1981 T h e t i m e - c o u r s e o f C a 2 + e x c h a n g e w i t h c a l m o d u l i n , t r o p o n i n , p a r v a l b u m i n a n d m y o s i n i n response to t r ans ien t i nc reases i n C a 2 + . B i o p h y s . J . , 3 4 : 5 5 9 - 5 6 9 . R o g e r s , S . L . 1982 M u s c l e s p i n d l e f o r m a t i o n a n d d i f f e r e n t i a t i o n i n r e g e n e r a t i n g m u s c l e g ra f t s . D e v . B i o l . , 9 4 : 2 6 5 - 2 8 3 . R o g e r s , S . L . , C a r l s o n , B . M . 1981 A q u a n t i t a t i v e assesment o f m u s c l e s p i n d l e f o r m a t i o n i n r e i n n e r v a t e d a n d n o n - r e i n n e r v a t e d g ra f t s o f the rat e x t e n s o r d i g i t o r u m l ongus m u s c l e . N e u r o s c i e n c e , 6 : 8 7 - 9 4 . S a i t o , A . , L a w , P . K . , F l e i s c h e r , S .1983 S t u d y o f n e u r o t r o p h i s m i n n o r m a l / d y s t r o p h i c p a r a b i o t i c m i c e . M u s c l e N e r v e , 6 : 1 4 - 2 8 . S a m a h a , F . J . 1972 T r o p o m y o s i n a n d t r o p o n i n i n n o r m a l a n d d y s t r o p h i c h u m a n m u s c l e . A r c h . N e u r o l . , 2 6 : 5 4 7 - 5 5 0 . 175 S a m a h a , F . J . , T h i e s , W . H . 1979 M y o s i n l i g h t c h a i n s i n D u c h e n n e d y s t r o p h y a n d p a r a p l e g i c m u s c l e . N e u r o l o g y ( N Y ) , 2 9 : 1 2 2 - 1 2 5 . S c h i a f f i n o , S . , G o r z a , L . , D o n e s , I., C o r n e l i o , F . , S a r t o r e , S. 1986 F e t a l m y o s i n i m m u n o r e a c t i v i t y i n h u m a n d y s t r o p h i c m u s c l e . M u s c l e N e r v e , 9 : 5 1 - 5 8 . S c h n e e b e r g e r , P . R . , N o r m a n , A . W . , H e i z m a n n , C . W . 1985 P a r v a l b u m i n a n d v i t a m i n D - d e p e n d e n t c a l c i u m - b i n d i n g p r o t e i n ( M r 28 ,000 ) : C o m p a r i s o n o f t h e i r l o c a l i z a t i o n i n the c e r e b e l l u m o f n o r m a l a n d r a c h i t i c ra ts . N e u r o s c i . L e t t e r s , 59: 9 7 - 1 0 3 . S i l v e r m a n , H . , A t w o o d , H . L . 1980 Inc rease i n o x i d a t i v e c a p a c i t y i n m u s c l e f i b e r s i n d y s t r o p h i c m i c e a n d c o r r e l a t i o n w i t h o v e r a c t i v i t y i n these f i b e r s . E x p . N e u r o l . , 6 8 : 9 7 - 1 1 3 . S m i t h , S . J . , W o l e d g e , R . C . 1985 T h e r m o d y n a m i c ana l ys i s o f c a l c i u m b i n d i n g to f r o g p a r v a l b u m i n . J . M u s c l e R e s . C e l l M o t i l . , 6 : 6 5 7 - 7 6 8 . S o u k u p , T . , V y d r a , J . , C e r n y M . 1979 C h a n g e s i n A T P a s e a n d S D H reac t i ons o f the rat e x t r a f u s a l a n d i n t r a f u s a l m u s c l e f i b e r s a f t e r p r e i n c u b a t i o n s at d i f f e r e n t p H . H i s t o c h e m i s t r y , 6 0 : 7 1 - 8 4 . S o u k u p , T . 1976 I n t r a f u s a l f i b e r types i n ra t l i m b m u s c l e s p i n d l e s . M o r p h o l o g i c a l a n d h i s t o c h e m i c a l c h a r a c t e r i s t i c s . H i s t o c h e m i s t r y , 4 7 : 4 3 - 5 7 . S r e t e r , F . A . , I k e m o t o , N . , G e r g e l y , J . 1966 S tud ies o n the f r a g m e n t e d s a r c o p l a s m i c r e t i c u l u m o f n o r m a l a n d d y s t r o p h i c m o u s e m u s c l e . In : E x p l o r a t o r y C o n c e p t s i n M u s c u l a r D y s t r o p h y a n d R e l a t e d D i s o r d e r s . A . T . M i l h o r a t , e d . E x e r p t a M e d i c a F o u n d a t i o n , N e w Y o r k , p p . 2 8 9 - 2 9 8 . S re te r , F . A . , S a l m o n s , S . , R o m a n u l , F . C . A . , G e r g e l y , J . 1973 S y n t h e s i s b y fas t m u s c l e o f m y o s i n l i g h t c h a i n s c h a r a c t e r i s t i c o f s l o w - t w i t c h m u s c l e i n response to l o n g - t e r m s t i m u l a t i o n . N a t u r e N e w B i o l . , 2 4 1 : 1 7 - 1 9 . S t a r o n , R . S . , P e t t e , D . 1986 C o r r e l a t i o n b e t w e e n m y o f i b r i l l a r A T P a s e a c t i v i t y a n d m y o s i n h e a v y c h a i n c o m p o s i t i o n i n r a b b i t m u s c l e f i b e r s . H i s t o c h e m i s t r y , 8 6 : 1 9 - 2 3 . S t e r n b e r g e r , L . A . 1979 I m m u n o c y t o c h e m i s t r y . L . A . S t e r n b e r g e r , e d . J o h n W i l e y , N e w Y o r k . S t i c h e l , C C , K a g i , U . , H e i z m a n n , C . W . 1986 P a r v a l b u m i n i n ca t b r a i n : I s o l a t i o n , c h a r a c t e r i z a t i o n , a n d l o c a l i z a t i o n . J . N e u r o c h e m . , 4 7 : 4 6 - 5 3 . S t r e h l e r , E . E . , O p p e n b e r g e r , H . M . , H e i z m a n n , C . W . 1977 I so l a t i on a n d c h a r a c t e r i z a t i o n o f p a r v a l b u m i n f r o m c h i c k e n leg m u s c l e . F E B S L e t t . , 7 8 : 1 2 7 - 1 3 3 . 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W . 1985 U l t r a s t r u c t u r a l l o c a l i z a t i o n o f the c a l c i u m - b i n d i n g p r o t e i n p a r v a l b u m i n i n n e u r o n s o f the s o n g s y s t e m o f the z e b r a f i n c h , P o e p h i l a gu t ta ta . C e l l T i s s u e R e s . , 2 4 1 : 7 7 - 8 3 . 178

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