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Alterations in fast and slow-twitch muscles of genetically dystrophic mice with special reference to… Johnson, Marjorie Isabelle 1987

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ALTERATIONS IN FAST A N D SLOW-TWITCH MUSCLES OF G E N E T I C A L L Y DYSTROPHIC MICE WITH SPECIAL REFERENCE TO PARVALBUMIN By MARJORIE ISABELLE JOHNSON B.Sc, The University of Waterloo, 1978. A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF T H E REQUIREMENTS FOR T H E DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Anatomy)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September 1987 © Marjorie Isabelle Johnson, 1987.  In  presenting  degree freely  at  this  the  available  copying  of  department publication  of  in  partial  fulfilment  of  the  University  of  British  Columbia,  I  agree  for  this or  thesis  reference  thesis by  this  for  his thesis  and  scholarly  or for  her  Department  V6T  DE-6(3/81)  1Y3  Columbia  I  further  purposes  gain  shall  that  agree  may  representatives.  financial  permission.  T h e U n i v e r s i t y o f British 1956 M a i n Mall Vancouver, Canada  study.  requirements  It not  be  that  the  Library  permission  granted  is  by  understood be  for  allowed  an  advanced  shall for  the that  without  make  it  extensive  head  of  copying my  my or  written  ABSTRACT  Muscular and  biochemical  progressive muscle.  fibers  1  parameters.  the  second  calcium-binding  alterations  slightly  increased in the  the  in  ATPase  histochemistry.  concentration  PV  samples.  significantly  and  in  the  content  suggests muscular  an  of  dystrophy  in and  that  the  between  alteration  differentiation  in  samples of PV  and  examine of  spindles  (EDL)  of  and fibers,  the  skeletal  and  their  genetically  ultrastructural muscle  histologically  the  the  spindles  unaffected  differences  findings  isolated a  2-week-old  the  were  was the  in  and  dystrophic  hypothesis  process o f skeletal muscle.  ii  dramatically  phenotypic that  between  similarity m u s c l e s at  strains  routine  were  myosin the  confirmed  the  in  No in  but  and  reduced  soleus.  by  EDL  gels  results  a  parvalbumin-  SDS-PAGE  These  The  of  by  (PV),  mapped  dystrophic  supported  dystrophic  muscles. slow  adult  on  RIA.  was  number  No  content  distribution  The  parvalbumin  mice  soleus.  increased in  supports  dystrophic  the  was  and  of  in  by  PV  fast  concentration  reduced  These  estimated  dystrophic detected  longus  physiology  process  muscle  extrafusal  procedures.  Parvalbumin was  and  2-week-old  dystrophic  data  and  and  significantly  immunohistochemical EDL  d e s i g n e d to  quantitative,  enzymatically  biochemical  adult  2-week  of  of  digitorum  surrounding  distribution  32  was  observed  the  morphology,  differentiation  histochemical,  in  the  in  and  fibers  the  was  development  extensor  appeared  chapter  immunohistochemical  on  study  the  dystrophy.  protein,  immunoreactive  and  affects  This  neonatal  to  fibers  fiber.  dystrophy  the  according  intrafusal  disease w h i c h  muscle  soleus  i n c i p i e n t stages o f m u r i n e In  the  muscular  the  Despite  their  genetic  examines  in  mice  a  of  of  Chapter  dystrophic  is  nature  effects  intrafusal  and  dystrophy  the  adult  changes  the  32  distribution  weeks  expression  of  dystrophy  alters  were  fiber the  of  types  age in  normal  TABLE OF  CONTENTS  Page Abstract  ii  Table of Contents  iii  List of Tables  vi  List of Figures  vii  Acknowledgement  x  Dedication  xi  General Introduction  1  Animal Models  1  i)  Histological Features  2  ii)  Fiber Types  4  iii) Physiological and Biochemical Changes  6  Statement o f the O b j e c t i v e s  Chapter  1  9  Postnatal development of muscle spindles in dystrophic  genetically  mice.  Introduction  10  Material and Methods  12  Histochemistry  12  Quantitation  13  Electron Microscopy  13  Observations  15  Histochemistry  15  M o r p h o l o g y and ultrastructure  18  Quantitation and distribution  25  Discussion  35 iii  Spindles in slow-twitch versus fast-twitch muscles  38  Morphogenesis  40  Functional and clinical considerations  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  Determination of Parvalbumin Content i)  77  Protein Determination  77  ii) R a d i o i m m u n o a s s a y  77  Results  84  Characterization of Parvalbumin  84  Characterization of Antibody  93  Immunohistochemistry i)  Adult E D L  ii)  A d u l t Soleus  100 107 ,  113  iii) T w o week E D L  118  iv)  119  T w o w e e k soleus  Summary of Immunohistochemical  Results  124  Parvalbumin Content  132  Developmental Changes  133  Discussion  145  Immunohistochemistry  145  i) M u s c l e S p i n d l e s  148  Parvalbumin Content  150  General Discussion  157  Bibliography  165  v  LIST O F 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  LIST 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 intrafusal fibers i n n o r m a l soleus a n d E D L muscles at 1 w e e k o f a g e  17  3-4. M u s c l e spindle in normal 3 week old soleus, myosin A T P a s e acid and alkaline preincubation 5-6.  20  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 s o l e u s at 3 w e e k s o f a g e , m y o s i n A T P a s e acid and alkaline preincubation  7-8.  20  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 a g e , m y o s i n A T P a s e acid and alkaline preincubation  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 a g e , m y o s i n ATPase acid and alkaline preincubation  22  11.  Schematic diagram of intrafusal fiber staining pattern  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 soleus  27  13.  Electron micrograph of a juxtaequatorial spindle f r o m a 3-week d y s t r o p h i c soleus  14.  29  E l e c t r o n m i c r o g r a p h of an equatorial spindle f r o m a 3 - w e e k dystrophic E D L  15.  29  L o n g i t u d i n a l a n d transverse reconstruction of spindle  distribution  i n n o r m a l soleus a n d E D L muscles 16.  33  L o n g i t u d i n a l and transverse reconstruction of spindle  distribution  i n d y s t r o p h i c soleus a n d E D L muscles 17.  35  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  of  parvalbumin  51  18.  General methodology outline  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  vii  21.  Standard  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 s a m p l e s  22.  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 s a m p l e s  81  23.  C o m i g r a t i o n of p a r v a l b u m i n w i t h E D L samples  86  24.  C o m i g r a t i o n of p a r v a l b u m i n w i t h soleus samples  88  25.  Isoelectric 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  adult  samples 26.  79  90  I s o e l e c 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-week  samples  92  27.  Two-dimensional P A G E of 2-week normal E D L  95  28.  Two-dimensional P A G E of 2-week dystrophic E D L  95  29.  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 soleus  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 soleus  97  31.  O n e - d i m e n s i o n a l P A G E used for immunoblotting  99  32.  I m m u n o b l o t o f 32 week o l d muscle samples  99  33.  Standard curve for R I A of cross-reactivity  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  35.  O n e - d i m e n s i o n a l P A G E and i m m u n o b l o t of 2 - w e e k - o l d muscle samples  106  36.  Immunohistochemistry  109  37.  Parvalbumin distribution in dystrophic 32-week-old E D L  109  38.  M y o s i n ATPase of normal 32-week E D L  112  39.  M y o s i n A T P a s e of dystrophic 32-week 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 soleus  115  41.  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 soleus  115  42.  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 soleus  117  43.  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 soleus  117  44.  Parvalbumin distribution in normal 2-week E D L  121  45.  Parvalbumin distribution in dystrophic 2-week E D L  121  46.  M y o s i n ATPase of normal 2-week E D L  123  of parvalbumin in normal 32-week-old E D L  viii  47.  M y o s i n ATPase of dystrophic 2-week E D L  123  48.  P a r v a l b u m i n localization i n n o r m a l 2 - w e e k soleus  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 soleus  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 soleus  128  51.  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 soleus  128  52.  Standard curve for R I A o f adult muscle samples  135  53.  Standard curve for R I A of neonatal muscle samples  137  54.  H i s t o g r a m o f R I A results f o r adult samples  139  55.  H i s t o g r a m o f R I A results o f neonatal samples  141  ix  ACKNOWLEDGEMENT  A  study  to  whom  my  s u c h as t h i s I  owe  advisor  Dr.  all-round  and  Baimbridge tried  Vogl, with  and  to  Dr. my  tribute  William while  who me  problems  to  her  owe  studies.  under  am the  for  to  the  deal  very  to  department last,  but  I  who above  I  the  and  would  endless  wish have all  Dow,  of  I  also  the  late  science.  of  I  Dr.  work  my  in  possible  to  have  Dr.  laboratory, Dr.  Wayne  there this  to  guidance,  his  being  would  people  indebted  committee,  always  she  best  liberal  Jasch I offer  hope  many  appreciation  the  deeply  to  for  my  patience,  me  you  have  receive  his am  truly  extend I  members  thank  To  technical of  ovarian  also l i k e patience  to  thank  made not  the  these  McLeod  thank  have for  assisted  her  and  for  work  A  me  to  help  work  as a  approved  special  throughout  with  introducing  Hella Prochazska,  instruction.  of  the me  Sue F i n l e y thanks  goes  my  animals, to  several  and  Bruce  to  Bernie  prints.  colleagues years  least,  who  transplants,  to  and  my  staff  Jean  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 Finally,  for  I  to  that  allowed  other  trivial.  appreciative  amputations  their  wish  him  wing,  the  Dr.  truths  I  education.  his  To  how  person, and  determined  thank  my  to  one  work.  great  techniques.  Anderson  matter  devotion  a  I  performing valuble  me  I  in  a scientist.  no  was  UBC.  interest  of  and foremost  who  Joanne E m e r m a n , and  this e x t e n s i o n o f her I  at  took  work  First  Ovalle  devoted  make  the  gratitude.  education  persistence, Ken  my  is n e v e r  I  of thank  Johnson.  x  and  study my  peers, a  truly  best  both  in  enjoyable  friend  and  and  outside  of  the  experience.  And  biggest  Paul  fan,  DEDICATION  To Auntie Bee, w h o s p a r k e d m y interest i n the w o r l d o f science a n d i n the u n k n o w n .  xi  GENERAL  Muscular  dystrophy  are  characterized  The  disease  affects  fiber.  Although  muscle  tissue  recent  isolation  dystrophy  by  progressive the  and  product  a candidate  appears  to  the  support  have  the  and  mutant  reported gene  for  theory  that  of  genetic  disorders  which  skeletal  muscle  fibers.  of  muscle  biochemical  been  cDNA's  the  of  degeneration  physiology  of  for  describe a group  severe  abnormalities  primary  of  u s e d to  morphology,  numerous  the  gene  is a t e r m  INTRODUCTION  in  has  both  not  a portion the  nature  gene  muscle  been  of  the  in  this  the and  non-  identified.  The  Duchenne  muscular  particular  disorder  is i n v o l v e d i n t h e 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 . , 1 9 8 6 ) .  Animal Models A  number  and  these  for  hereditary  model an  of  include  animal the  hamster,  muscular  completely  invaluable  models  chicken  dystrophy  resembles  wealth  of  the  dystrophy (Meier is  two are  and  and  carried  by  splaying a  the  experimental  an at  the  and  undergo  clenching ability severe  No  c l i n i c a l signs are  the  disease  is  to  expressed  the  atrophy  in  in a  in  muscular  In  this  be  considered.  form  of  muscular  to  dystrophy  thesis,  the  Even  knowledge  of  mouse  models  for  the  research,  murine  model  though  no  animal  they  have  added  dystrophy,  our  et  al.,  1955)  Muscular dystrophy gene are  (Harris  their  and  the milder  and  disease a n d  inability  balance.  become  forelimbs. form 1  than  in  mice  that  these  to  the  to  grasp the  two  early  in  mice  of  Mice  age  of  their  disease  homozygous  noted  strains  with  extended  muscular  C57BL/6Jdy  1980).  an  abnormally In  the  at  As  progressive  and  Slater,  characterized  hindlimbs,  regain  evident  mouse.  (Michelson  allele  of  utilized  only  studied  recessive  dystrophic  and  data  strains.  autosomal  decreased  hindlimbs  frequently  1970)  been  environment.  129/ReJdy/dy  Southard,  homogeneous by  most  will  human  response o f muscle i n an a b n o r m a l The  have  and  2 J  /dy  2 J  mouse  that  2-3  are  weeks  hind  feet,  progresses,  the  dysfunctional.  for  the  of  the  dy  2 J  dy  allele allele.  Mice  of  muscle  the  dy  tissue.  myotubes  strain  2 J  The  are  disease  Recently  Bulfield  et  (mdx).  Although  serum  enzyme  levels  subsequently  been  The  mdx  shows  mouse  a  to  3-4  prolonged  al.  a  slower,  appear  to  affect  and  a  (1984)  genetic to  only  a  adult  mouse  type  differentiation  homology  characterized signs  weeks  shows  of  of  no  and  Therefore,  described  Duchenne  few  time  signs  of  to  its  progressive the  degeneration  myogenic  myofibers  X-linked  the  human have  potential  are  process  altered  fiber  recessive locus  degeneration  of  since  at  sustained  muscle  the  the  birth  regenerative  the  and  this  high  mutant  muscle  regeneration.  and  altered  physiological  tension, muscle animal.  weakness,  no  parameters  capacity  exist,  in  for  control  physiological  mutant  reported,  is a d e c r e a s e d t w i t c h to  may  been  model  compared  of  high  to  a  There  measures  due  as  an  dystrophy  muscle  age.  half-relaxation  mdx  normal.  not  normal  similar  has  and  does  by  1975).  mouse  up  characterized  morphologically  (Platzer and P o w e l l ,  responses  are  the  weight  However, changes  have  mdx  in  the fiber  returned  mutant  to  has  not  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 .  Histological Features Several of  the  histological  murine  development 1981b).  of  disease, indicating  (Platzer  The  appearance  observed  extrafusal  size  in  and  a  1980).  are  of  phases  of  the  fascicle  disorder  of  regeneration there  is  in  Mendell  dystrophic or  necrotic, to  postnatal  those  muscle  et  fibers  significant  al.,  Summers  1979; muscle of  regenerating  and  a is  the or  normal  proliferation  early  the  muscle Parsons,  heterogeneity  disease, muscle splitting  in  the  1981b). of  stages  in  and  are  appearance  common  Parsons,  is  t i s s u e 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 . , 1 9 7 9 ) .  2  pre-clinical  occurs  stages  with  and  expression  mouse  later  (Summers a  the  phenotypic  early  are  next  Fragmentation  abortive  of  At  that  its  1975;  feature  fibers.  occur  that  Powell,  those  given  Mastaglia, signs  and  characteristic  of  variable  abnormalities  fibers  commonly  (Cullen  initial During  endomysial  in  and  stages, the  as  later  connective  A  characteristic  regenerative 1979;  activity  Summers  at  and  of  1981a).  Myosatellite  it  the  initial in  myosatellite  dystrophic  is  mouse  formation.  suggested  to  fuse  to  the  This  the  murine  events  form  of  new  adequate  development  of  the  decreases  responsible  cell  for  has  the  of  et  inability  of  (Banker  of  et  al.,  abnormal  and  Parsons,  investigators  reported  cells  and,  abnormal  and  in  dystrophic  1984).  the  to  (Summers  is  al.,  presence  an  satellite  higher  (Ontell  the  attributed  These  which is  is  necrosis  necrosis  number  myotubes  age  fiber been  fiber  population  with  of  myotubes.  an  myosatellite  dystrophy  area  feature  has  and  be  to  of  from  1981b).  muscle  The  muscle  feature  distance  cells  cells  subsequent  normal  some  Parsons,  response  that  histological  not  their  muscle  than  factor  was  This  regenerative  therefore,  response  of  muscle  is  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 t h e m u s c l e d e t e r i o r a t i o n . One  of  dilatation  of  cisternae and  the  first  the  SR,  (Platzer  and  including Powell,  filled  with  matrix  Retraction  clots  form  leading  coagulation  motor of  to  end-plates  both  neural  model. the  2 J  strain,  have  alterations,  retraction  of  described.  - As  been  reported  mutant early  failure in  and  the  of  postnatal  both the  network  and  sarcolemma and  (Banker  myotrophic  allelic  Schwann cell  development  al.,  influences (Saito  et  al., 3  1978).  appear  swollen  and  atrophied.  fragmented,  eventually  disease  nerve  al.,  of  have the  1983). of  number  and  ensheath the  may  indicated  the  axons of  This  been  this  animal  observed  junctional  group  1978).  of  presence  degeneration  myelinated  Swenarchuk,  naked  the  in  membrane,  of  studies  of  Presynaptic  the  postsynaptic  (Montgomery  (Bray  have  atrophy  the  terminal  Ultrastructural  with on  et  the in  slightly  because  and  and  fragmented  1979)  associated  from  to  become  mouse  tubules  mitochondria  influences  reductions  strains  longitudinal  infiltration.  et  as s i m p l i f i c a t i o n terminals  dystrophic  becomes  phagocytic  mice  significant  of  myofibrils  documented  such  in  Subsequently,  neurotrophic  been  alterations  abnormalities  definite  nerve  well,  in  the  necrosis  abnormal  neurons  postsynaptic  granules,  muscular  no  its  1975).  dystrophic  and  Although  dy  motor  in  ultrastructural  folds  in the and and  have  been  axons  have  In  the  s p i n a l roots concluded  dy  2 J  occurs  that  the  Schwann  cell  impairment laminas  deficit  of  of  was  Schwann  Schwann  a  developmental  cell  multiplication.  cells  is  a  common  abnormality  associated  In  discontinuities  addition,  abnormality  observed  with  in  a  neonatal  in  the  the  dy  basal adult  2 J  d y s t r o p h i c m o u s e ( M a d r i d et a l . , 1 9 7 5 ) .  Fiber Types  type  Extrafusal  muscle  1  according  (slow),  Kaiser,  1970;  activity  (pH  Peter 9.4),  (Brooke  contraction  times,  fibers type a  and are  2  a  also  Type  2  basis  of  fibers  differential  values (Brooke  of  type  2B,  Generally, 2B  of  combined  2A  Kaiser,  1970).  These  oxidative  high  into  Kaiser, type  fibers  systems  in  et  et  Type are  al.,  referring  stable  at  acid  It  according The  does  not  two  staining  high  al.,  always  fibers  these  oxidative 2A,  2B,  such  type  times,  and  fibers  are  enzyme  activity.  and  on  2C)  buffers  at  acid-labile than  and  alkaline  apply  to  more as  while  type  oxidative  and  are  type  often 2A  appropriate  type  fibers  preincubation.  (FOG)  their  the  varying  more  to  1  Conversely,  contraction hand,  slow  mitochondrial  1972).  nomenclatures  is, therefore,  extrafusal  4  histochemical  fast-oxidative-glycolytic  1972).  2A/FG.  are  and  ATPase  preincubation  fibers  (FG)  1982). to  with  (Brooke  Consequently,  other  (types  and  myosin  a  fast  poor  2A  termed  correlation al.,  and  treatment  fast-glycolytic  this  species ( G r e e n  to  also  (Peter  the  several subgroups  fibers  are  activity,  (fast)  associated w i t h  activities.  On  content  low  system,  et  2  stains  have  usually  (Peter  ATPase  system.  1970).  2C  are  enzyme  type  enzyme  conventional  fibers  fibers  types,  fibers  enzyme  (SO)  myosin  enzyme  divided  enzyme  terminology  for  to  glycolytic  mitochondrial  designated  all  developed  have  however,  muscle  according  are  interchangeably; fibers  and  1  major  glycolytic  stable  susceptibility  whereas  type  fibers  glycolytic  be  Type  two  or  acid  low  may  pH  1972).  glycolytic a  into  ATPase  slow-oxidative  fibers  by  classified  myosin  potential  termed  developed  characterized  are  poorly  high  are  to  al.,  and a  extrafusal  well  et  but  procedures  content,  fibers  2A/FOG  used  and to or  2B  utilize type  Fiber of  type  dystrophic  involved  in  severely  of  of  Dribin  and  Ovalle  et  changes  al.,  1983;  of  increase  in  type  fibers  have  fibers  and  al.,  complemented adult  et  by  fiber  types and  human  muscle  (Bandman,  resembling  a  2C  type  fiber  or  the  and  advancement murine  that  point  (Jasch to  the  indicate  an  is  et  fiber  al., in  in  also  and of  the  1982)  in  during  point  similar  a fetal and  fiber  to  a  muscles. mice,  the  This  1981)  the  muscle  an  type  1/SO  of  FOG  percentage  data  extrafusal  alteration  normal  fast-twitch  supported  Hoh,  their  1983)  decrease i n  these  immature  dystrophic  disease.  al.,  in  mouse  et  that  with  retardation  appearance of  Conversion  suggested  histochemically  dystrophy  histochemical  1981;  Both  the  of  Parslow,  soleus  of  (Fitzsimons  1985). may  are  number  and  Wirtz  1983).  more  Parry  2 / F G variety  al.,  be  A the  muscles  1977;  of  a concomitant  which  affect  been  a  to  1977).  dystrophic  1981;  type  et  fibers  in  to  preferentially  Cosmos,  type  due  strains  and  recently  slow-twitch  (Ovalle  muscle  dystrophic  has  dy  (Butler  1980;  Studies of  biochemical  investigations  dystrophic  of  fibers  with  muscle  are  thought  Simpson, can  two  1977), whereas  are  dystrophy  Parslow,  the  atypical)  increase  1983)  and  of  and  the  muscles  fibers  'intermediate'  a l . , 1983).  reported  (or  It  between  Cosmos,  Atwood,  1983),  fibers  and  skeletal  and  al.,  extrafusal  been  in  an  et  differ  fast-twitch  (Dribin  1983).  to  extrafusal  abnormal  et  types  al.,  (Ovalle  the  muscular  Silverman et  to  oxidative  strain  2 J  fiber  development  also  strain,  murine  (Wirtz  2/FOG  reported  disease (Butler  disease ( P a r r y  extrafusal  (Bressler  in  the  significantly in  the  conversion  decrease i n postnatal  that  Wirtz  properties  During  trend  a  dy/dy  /dy  2 J  1977;  development  dramatic  dy  been  slow-twitch  extrafusal  Simpson,  stages  fibers,  the  evidence  involve  postnatal  the  phases o f  in  the  histochemical  has  predominantly  provide  character  In  eariy  affected  reports  early  mouse.  the  composed  susceptibility  to  type  2C  cytochemical  by  physiological  and  is  further  form  of  myosin  adult types  maturational  chicken to  those  process  of  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 ( O v a l l e et a l . , 1 9 8 3 ; W i r t z et a l . , 1 9 8 3 ) . Less of  is  intrafusal  known fibers  about in  the  the  effects  muscle  of  dystrophy  spindle.  on  the  histochemical  These specialized and 5  partially  development encapsulated  sensory  stretch  receptors  appear  Morphologically  abnormal  intrafusal  dystrophy murine  (Swash  and  dystrophy  are  intrafusal report  fiber  spindle.  In  throughout this  by the  type  murine  addition,  are  has  muscular  length  of  not  been  considered.  ATPase  of  necessary  course  These  staining  in  in  a  intrafusal  an  individual  spindle  to  determine  the  requires  (Soukup,  response,  in  either  in  dealt  with  type  serial  of  in  either  the  of  the  development of  in  intrafusal  sections  further  any,  recent  region  identification that  A  morphology  confined  1976),  if  spindles  1974b).  spindle  fiber  Since accurate  methods  muscle  (Yellin,  1982).  Duchenne  abnormalities  studies  fibers  (Swash,  human  of  any  alterations  of  in  studies  histochemistry some  disease  noted  detected  intrafusal  time  the  been  dystrophy.  the  normal  or  shown  to  microscope not  1969)  has  the  myosin  have  (Meier, 1986)  have  Light  and  histochemistry  muscle  types  Dow,  of  or  dystrophic fiber  and  stages  morphology  limited  resistant  fibers  1976).  morphology  (Ovalle  terminal  Fox,  relatively  be  cut  investigations  intrafusal  fibers  of to  dystrophy.  Physiological and Biochemical Changes The  results  dystrophy  are  decrease peak  in  and  studies  consistent  both  tension  (Parslow  of  twitch  and  a  Parry,  from  32 weeks o f  in  the  was  prolonged correlate seen  in  significantly  these  the  mechanical  the  histochemical  tetanic  force,  increase  al.,  1983;  and  and  tension  presence  of  not  in  tetanus  the the  slow (Parry  normal  dystrophic myosin, and 6  Generally,  time  and  tension  a l . , 1983).  half-relaxation  from  Parry  time  in  values  by  has  Desypris,  of  1983).  and  1983).  observed  young  muscle  of  age.  has  been  a  muscle For in  the mice  observed soleus  While found  relaxation changes  is  time-to-  dystrophic  twitch These  murine  there  prolongation  32 weeks  prolonged  in  fast-twitch  been the  the  fast-twitch  time  Desypris,  However,  the  muscle  contraction  B r e s s l e r et twitch  skeletal  findings.  half-relaxation  a g e ( B r e s s l e r et  has  an  of  the  different  muscles  properties  in  tension  time-to-peak with  and  decreased  time-to-peak  not  with  1981;  soleus,  to  the  prolongation  dystrophic 4  of  the to  phase  suggest  a  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 Wood  et  al.,  phosphoenzyme  1978),  a  2 +  transport (Sreter et al., 1966; Mrak and Baskin, decrease  in  maximum  steady-state  intermediate, and a decrease in Ca -sensitive 2+  and Baskin, 1978; Martonosi, 1982).  1978;  concentration  of  ATPase activity (Mrak  In addition, densitometric analysis of dystrophic  SR preparations indicate a decrease in calsequestrin in the dy  2J  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). calcium  is  transmission, intracellular  mediated  by  reception milieu.  certain  and The  proteins  that  removal,  thereby  roles  calcium,  of  interact  with  controlling and  its  its  the  Regulation of  ion governing  concentration  affiliated  binding  in  its the  proteins  (calmodulin, myosin phosphorylated light-chain, parvalbumin, and troponin C) in muscle contraction have 1985).  been  investigated  extensively (reviewed  by Carafoli  and Penniston,  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 7  muscle  fibers of  dystrophic possibly the be  mice  due  calcium  lies  initiation muscle,  an  the  in in  of  the  a  topic  muscle  contraction In  al.,  of  concentration  the  Ca  2 +  PV  affects  decreased, calcium et  al.,  1984).  during  the  proposed  -regulated  of  PV  stages  Ovalle, the  and  the  1986).  protein  may  be  some  disease  (Jasch  extent of  to  of  that  the  not  the  significance  occurring  which Ca  has  during  Stuhlfauth  et  al.,  muscles  Moase,  intracellular  may  fast-twitch  fast-twitch and  SR  Hence, P V  those 1982;  though  and  in  1985).  as  of  capacity  phase PV  al.,  the  indicate  of  et  Even  from  would  Heizmann,  The  1978).  relaxation  (Klug  concentration,  2 +  functions  this  content  of  buffering  the  Ca  release  This  processes s u c h  (Celio  the  later  and  sarcolemma (Duncan,  diseased muscle  dystrophy,  Johnson of  the  myopathies  in  the  capacity  and  reduced  1985;  functional  sarcoplasmic  system  of  in  in  elevated  (Volpe  light  in  2 +  an  S R has  interest  murine  significantly  the  Ca  in  regulation In  in  of  the  dystrophy  calcium  alteration  to  changes  of  contraction.  become  et  linked  ability  murine  regulation  1984).  been  permeability  uptake  unaffected  problem  in  to  has  1985;  is  Pette  this  decreased  in  dystrophic  2 +  m u s c l e is u n c l e a r . The an  etiology  altered  tracers  et  Leakage  suggesting  the  in  1979;  that  focal  cellular  internal  ultrastructural freeze-fracture  of  muscular  structure  penetrate  al.,  disrupted.  event  murine  membrane  focally  (Banker  of  the  Mendell  et  calcium  into in  degeneration  studies images  the  have  al.,  the  of SR  been  of  unknown, been  muscle  fibers  fibers  1979).  not  as  dystrophy  well.  dilatation  to  cellular  and  is  necrosis  the  initial  apply  addition  and  muscle  integrity  properties In  (Platzer  SR  murine  represent  Abnormal  in  showing  early  pointing  Extracellular  membrane  during do  muscle  mouse  evidence  dystrophic  that  occurs  dystrophic  published  in  membrane  al.,  but  accumulating.  indicating  plasma et  is  has  1979)  muscle  (Mendell  systems of  function  sarcolemma  defects  membrane  or  dystrophy  to  Powell,  to the  1975)  fragmentation  w i t h n o a b n o r m a l i t i e s o f the transverse tubules ( M r a k a n d B a s k i n , 1978). A dystrophic  correlation muscle  of is  the  difficult  morphological, because  of  a 8  biochemical combination  and of  physiological  factors  that  may  data  on  contribute  to  false  in  impressions.  fiber  particular hindered  the  type  composition,  stage  of  by  protease  the  possibility to  the  the  species  disease.  data  release  of  of  observed  some  unexposed  increased  the  of  lysosomal  defect  project  into  be  and  fatty  changes  due  to  the  tissue  are  connective-tissue,  preparation.  alterations  considered  by  homogenized  during  biochemical  limited  problems  of  fibrous  enzymes  must  and  sampling  preparations  amounts  primary  complicated  and  Biochemical  and  the  is  variations,  of  that  an  of  presence  activity,  reactions  Interpretation  could  As  be  (Martonosi,  well,  secondary  1982;  Engel,  1986).  Statement o f the O b j e c t i v e s I  have  divided  morphological,  histochemical  intrafusal  fibers  muscular  dystrophy.  1986).  type of  in  Secondly,  certain  this  proteins  fast  and  time,  and  with  an  I  dystrophic  This  may  in also  muscle.  immunochemical at  an  review  of  the  was  be As  marker  earlier muscle  changes  by  the in  dy  responsible  could  I  be  than  spindle  and  for  a  wished used  to  myosin of  determine the  ATPase  parvalbumin  9  if  or  the  type  in  whether  expression of  given  in  the  are  An  calcium  histochemistry. is  Ovalle,  profile of  of  muscle response  half-relaxation  expression  the  and  the  to  their murine  physiological  respect  the  of  alteration  parvalbumin.  disturbance  detect  loss  in  study and  stages  (Johnson  differences a  to  spindles  neonatal  with  fiber  protein  to  corresponding chapters. .  that  reflect  extrafusal  wished  published  mouse,  2 J  I  muscle  the  determining  calcium-binding  well,  stage  in  been  muscle  in  during  hypothesis  attempted  muscle  the  recently  the  Firstly,  changes  muscles  dystrophic  accompanying  alteration  has  test  from  slow-twitch  fibers  work to  studies.  quantitative  slow-twitch  wished  isolated  the  parvalbumin  and This  differentiation. fast  and  two  associated  alteration homeostasis  in of  parvalbumin  as  dystrophy  muscle  A  in  more  introduction  an  extensive to  the  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  INTRODUCTION  Mammalian encapsulated types  and  muscle  intrafusal have  morphological  spindles  muscle  been  are  fibers.  designated  (Banks  et  al.,  sensory  These  bag ,  bag  x  1977),  stretch  fibers  can  and  chain  2  histochemical  receptors  be  differentiated  fibers  (Ovalle  that  contain into  three  to  their  according  and  Smith,  1972),  and  p h y s i o l o g i c a l ( K e n n e d y et a l . , 1 9 8 0 ; B o y d a n d S m i t h , 1 9 8 4 ) f e a t u r e s . In vary  contrast  to  histochemically  Kucera,  1977;  their  neighbouring  along  their  Kucera  Immunohistochemical fibers  differs  between  individual  and  studies  extrafusal lengths  Dorovini-zis,  indicate  bag and  fibers,  that  chain fibers  mammalian  (Yellin, 1979;  the  myosin  1974a;  Banks  Soukup  et  et  composition  (Pierobon Bormioli  intrafusal  et  al.,  1977;  al.,  of  al.,  fibers  the  1979;). intrafusal  1 9 8 0 ; et  Kronnie  et a l . , 1 9 8 1 ) . Unlike and  Mastaglia,  their  1975;  James  abnormal  Meek,  disease. the  time  course  of  murine  appear  al.,  which (Law  to  be  fibers  1977)  1979)  have  studies  fibers  in  intrafusal  a  morphologically  al.,  1983)  been  Duchenne  failed  in to  type  with  region  altered  muscular to  the  reported  in  myotonia  dystrophy  any  the  development  in  the  dystrophic  spindles  (Swash,  Fox,  changes  In  (Cullen  Fox,  1976)  in  1974b; with  histochemistry  addition, muscle  1982).  associated  or  and  and  1969; Y e l l i n ,  morphology  spindle.  human  (Swash  and  (Meier,  pathological  either of  disease  (Swash  dystrophy  in  dystrophy,  resistant  murine show  dealt  confined  fiber  et  have  and  either  are  relatively  spindle morphology  These  intrafusal  fibers,  intrafusal  et  studies of  and  and  fibers  Maynard,  humans,  the  extrafusal  1980)  intrafusal  Although  of  the  has  the not  normal been  considered. Despite concerning  the  the  extensive  early  literature  development  of  published the  disease  10  on  muscular  with  respect  dystrophy,  information  to  spindles  muscle  is  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  MATERIALS A N D  Normal the  mouse  the  maintained  and  dystrophic  ME.  weeks.  time  and  colony  normal  Harbour, 3  C57BL/6J(+/+)  Male  When  as t h e i r  dystrophic  in  our  possible,  dystrophic  C57BL/6J(dy  department.  strain  homozygous  were  were  controls  counterparts.  A  The  obtained  offspring  normal  METHODS  of  /dy  2 J  original  from  s e l e c t e d at similar  total of  28  )  2 J  ages  mice  were  breeding  housed of  both  Jackson  Laboratories,  Bar  postnatal  ages o f  and  were  pairs  in  sampled  muscles f r o m  1, 2 ,  at  the  14 n o r m a l  same  mice  and  3 0 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 .  Histochemistry Animals extensor and  were  digitorum  longus  quickly  excised.  then  physiological in  fresh  saline  mouse  orientation. frozen  on and  chain  intrafusal  At  (pH  9.4)  over 40x  the  their  for  from  origin  100pm, myosin  length full  objective.  was  cryostat  Intrafusal and  established  The  insertion,  consecutive  acid fibers  Smith,  and  12  the  typed  1972).  throughout  at  Brooke,  alkaline  were  and  The its  were  by  embedded for  future  tragacanth  and  quickly  were  sections  were  (pH  1973).  of  and  on  mounted 4.6)  sequence  was  using the profile  for  were  pH  muscle  to  histochemical except  were  This  to  collected  4.2  preincubations according  allowed Muscles  sections  distribution  length,  removed  moistened  sectioning.  acid  right  tendons  chucks  transverse  histochemistry and  to  gauze  the  gum  The  fascia  Muscles  of  using  prior  each muscle, and at  superficial  surgical  location  hour  (Dubowitz  decapitation.  measured.  chucks  to  ATPase  extent  (Ovalle  then  of  on  nitrogen. 1  three  placed  the  on  liquid  preincubations  classification fiber  mounted  or  exposed,  were  note  with  thick)  for  to  were  was  weights  care  dislocation  soleus  muscle  muscle  the  every  cervical  and  Each  wet  in  (10pm  throughout  mapped  (EDL)  cooled  coverslips  alkaline  microscope and  -20°C  coverslips.  separate  either  specimens were  sectioned  followed was  to  by  taking  isopentane  equilibrate  glass  and  liver,  The  in  serially  killed  the  spindles the  bag  l 5  for  light bag  2  each  equatorial  zone,  where  distinguish  little chain  diameters  and  however,  those  or  no  from  fiber  staining bag  fibers  lengths.  that  occurred. and  In. most  were  similar  In  addition,  bagj  from  bag  cases,  chain  fibers  in  size  to  morphological fibers  2  were  bag  criteria  included smaller  fibers  used  to  cross-sectional  than  exhibited  bag  the  fibers; staining  properties of smaller chain fibers.  Quantitation Muscle  spindles,  microscope cross of  at  section.  The  of  which  the  intrafusal  fibers  and  spindle  interval  length  in  myotendon  nonencapsulated  100pm  cross-sections  and  of  each  or  per  nerve  each  drawn  spindle  intrafusal  on  was  fibers  fibers,  approximated  by  were  The  seen.  and  the  location  were  noted  for  diagrammatic  The  muscle spindle  were  located  a replica sketch of  spindle  tissue  distribution.  and  intrafusal'  index  of  (number  muscle  counting  the  number  number  of  spindles  receptor  longitudinal of  the  entire  each  the  under  relative  to  reconstructions  muscle spindles per  gram  muscle weight) was calculated f o r each muscle e x a m i n e d .  Electron Microscopy Mice aqueous fixed  at  postnatal  sodium  with  rapidly  and  placed  in  dehydrated mixture. glass  Animals  in  1  with  graded  and and  week  a  0.1  old  lmg/gm  EDL  into  1 to  2 hrs  and  propylene of  anesthetized body  decapitated,  right  and  then  blocks  blue.  collected 13  on  and  buffer  hindlegs  soleus  were  Selected copper  grids,  7.3)  room  removed  and each  M u s c l e pieces  were  1% a q u e o u s  Os0 ,  in  of  at  was  from  an  each group areas  with  perfusion  dissected  with  embedded from  (pH were  segments.  2  postfixed  and  intraperitoneally  weight,  and  2 mm  oxide,  random  toluidine  sections  were  Sorensen's phosphate  were  sections (0.5-1.0pm)  ultrathin  M  blade  and  with  weeks  dosage o f  a further  ethanol  stained  3  The  a razor for  and  in  fixative.  fixative  Thick  knives  retrimmed,  in  minced  fresh  at  glutaraldehyde  immersed  animal  2  pentobarbital  2.5%  temperature.  ages  the  stained  4  Araldite/Epon were  cut  blocks with  with were uranyl  acetate  and  lead  citrate  and  examined  with  microscope.  14  a  Phillips  E M  200  transmission  electron  OBSERVATIONS  A  total  muscles each As  of  were  598  examined  muscle. in  muscle  Most  other  spindles  in  serial  spindles  species  (see  from  the  transverse  consisted of  review  by  normal  and  dystrophic  sections, f r o m  two  Boyd  bag  and  proximal  fibers  Smith,  EDL  and  to  usually  1984),  the  distal  two  bag  and  soleus  ends  chain  fibers  of  fibers.  extended  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 .  Histochemistry Intrafusal ATPase  (Figs.  Under  acid  were  usually  soleus  and  appeared  regions were  of  be  could  of  both  intrafusal  and  seen between  1,  and 2,  sections  acid  preincubation.  were  bag  stable  2  fibers,  and  under  both  treated  the  pH  acid  preincubation.  from  light  at  pH  to  fibers  were  lightly  fibers  2  be  was  seen b y  were  lightly  same  zone  staining  weeks  At  or  profile  was  week The  No  changes  of  However,  by  in in  fibers  t  stained.  of  age  in  of  age,  the as  This both  bag  bag  weeks  fibers  2  fiber  x  fiber  bag  was  polar typing  fibers  x  of  stained  spindle  intrafusal  the  pattern  intensely  muscle  2  spindles.  bag  staining  sometimes  myosin  muscle  darkly  noted  for  stained,  spindle,  muscles.  EDL  all  1 week 1  a  stained,  age.  in  stained. of  the  no  fibers. alkaline  alkaline bag  x  fiber  conditions,  after  acid  2  3  after  Under  only  This  in  types  genotype.  moderately  dystrophic  l o n g e r s t a i n e d as d a r k l y as b a g Serial  or  the  those  three  could of  to  into  bag  fibers  Within  1,2).  normal  and  fibers  chain  lightly  (Figs.  chain  regardless  variable.  similarly  differentiated  stained,  stained  either  be  stained  conditions,  1,2),  fiber  2  soleus  (Figs.  were  to  bag  and  profile  EDL  fibers  the  the  moderately  darkly  observed as  1,2)  of  preincubation  histochemical  bagj  fibers  The dark  conditions, was  lightly  whereas  chain at  preincubation  the  fibers  alkaline  fibers  stained. bag  x  underwent  pH. 15  chain  were  Thus  the  were  only  complete staining  to  those  darkly  Therefore,  showed a  compared  bag  stained fibers  2  moderate reversal  pattern  after  in  as  were  stability staining  exhibited  by  Figures 1,2 Transverse frozen sections of portions of a normal soleus (Fig. 1) and E D L (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 E D L . Bag (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. 2  16  the  chain  fibers  fibers  of  the  from  that  of  genotype,  resembled  type  2  the  variety  type  animal  the  2  age,  reversal  (Figs. 3-6).  or or  typical  type  In  contrast,  1 extrafusal  muscle  observed  fibers  phenotype,  in  the  the  bag  surrounding fiber  (Figs. 5-6).  this  pattern  extrafusal  reactivity  differed  Regardless of  remained  murine  consistent  (Figs  7-10). Myosin the  length  Figure  ATPase  of  11.  an  alkaline showed chain  and  bag  and  dual  fibers  were  at  lost  each  under  this  both  and  was  age  darkly  fiber  in  the  lightly pH  are  types  varied  summarized  extracapsular  polar  along  schematically region  toward  the  equatorial  throughout  its  length.  after  zone. The  bag  variations.  a l k a l i n e - s t a b l e , also w i t h  no  regional  variation.  studied,  and  it  intrafusal did  not  fibers differ  of  both  between  acid  fiber  2  regional  neonatal  in  Under  no  of  conditions,  slightly  with  typical  group  intrafusal  reactivity  stained  acid-labile  pattern  three  These variations  stained  fiber  reactivity  staining  EDL  this  the  spindle.  fiber  2  of  gradually  conditions a  overall  individual  The  preincubation,  reactivity  the  The This soleus  normal  and  dystrophic animals.  Morphology and Ultrastructure The  first  soleus  of  dystrophic  animals  unaffected  by  dystrophy  in  the  occurred  the  soleus  12).  obvious  Within  these  extrafusal  fibers  population  of  to  be  fiber  as  greater changes  in in  in  and the the  all  areas  at  3  the  ages  dystrophic  well  as  muscle  presence  dystrophic surrounding  cells ( F i g .  muscle,  than  age.  that  spindles 12).  extrafusal  muscles  of  muscles examined  in  spindles 18  The  rich  the  was  their and  the  with normal their  the  centrally  spindles  an  the  increased  fibers  appeared  variability  located  nuclei  fibers  Despite in  in  (Fig.  The  counterparts. intrafusal  alterations  with  in  appeared  surrounding  extrafusal muscle.  detected  however,  muscle  infiltrated  dystrophic  fibers  in  were  endomysium  Some of of  EDL,  Interestingly,  were  muscle,  changes typical of  the  studied.  muscles  of  tissue  in  weeks  neonatal  of  pathological the  changes  areas  connective  undergoing size  microscopic  in was  these  dystrophic  Figures  3-6  S e r i a l s e c t i o n s o f p a 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 ) s o l e u s at 3 weeks of age, stained for myosin ATPase after acid (left) and alkaline (right) preincubations. In both normal and dystrophic muscles, polar regions of spindles contain bag fibers (arrowheads) that stain m o d e r a t e l y at a c i d p H and lightly at alkaline p H . Bag f i b e r s (asterisks) react intensely u n d e r b o t h c o n d i t i o n s , whereas c h a i n f i b e r s ( a r r o w s ) 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 l s o 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 y p e s 1 (1) a n d 2 (2). B a r = 2 0 p m ; x 1,200. 1  2  19  20  Figures 7-10 A l t e r n a t e s e c t i o n s o f p a 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 , 1 0 ) E D L at 3 w e e k s o f a g e , 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 ( l e f t ) a n d p H 9.4 ( r i g h t ) . Bag fibers (asterisks) are highly reactive at both preincubations, whereas bag fibers (arrowheads) vary i n intensity of staining. C h a i n fibers (arrows) stain similarly 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. 2  x  21  21  Figure  11  Schematic diagram illustrating 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 properties o f the bag! (B ), bag ( B ) 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 neonatal m o u s e . Composite longitudinal reconstructions (above) were made f r o m transverse serial sections (below). 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 t h e e q u a t o r i a l zone. 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 o n e h a l f o f a spindle, comparing intrafusal f i b e r reactions after a c i d (left) a n d alkaline (right) preincubations. 1  2  2  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 t h o s e o f n o r m a l c o n t r o l s ( F i g . 1 2 ) . Ultrastructural spindles, two  where  spindles  each  case,  similar electron  in  and this  muscles  (Fig.  space, outer cells.  The  a  the  inner  were  the  inner  capsule  under  a  capsule  dystrophic  also  dystrophic both  of  the  composed  least In was  and  are  14  and  and  EDL,  dystrophic  was  enveloped  perineurial  epithelial  by  a  clear  perineurial of  At  animals  soleus  spindle  surrounded  compartments  13  normal  each  of  microscope.  Figures  Like  regions  13,14).  electron  lamellae  was  (Figs.  the  region,  fibers.  was  separate  in  of  concentric  capsular  the  3-week  equatorial  intrafusal  formed  distinguished  appearance  inner  intracapsular  counterparts.  from  of  the  spindles  normal  central  consisting  isolated  thin  easily  of  typical  the  to  examined  spindle  single-layered it  were  their  the  Within  A  confined  morphology  muscle  capsule  which  capsule,  of  age.  14).  from  group  of  represent  multilayered  cells  age  was  fibers  ultrastructural  that  at  chain  each  micrographs  spindles a  and  from  respectively,  by  bag  the  to  examination  cells  flattened,  around  the  fibers  was  periaxial of  the  contiguous  intrafusal  fibers  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). The  myofilament  equatorial formed  (Fig.  discrete  Satellite chain  zone  cells  fibers  situated  in  content 14).  (Fig. shallow  both  bag  Myofilaments  myofibrils,  were  of  often  found 13).  separated  beneath  the  Sensory  cavities  on  in  the by  chain  juxtaequatorial mitochondria  external  terminals the  and  lamina  abundant  intrafusal  fiber  portions  of  of  varying  associated with  reduced  sizes  with  (Fig.  14)  (Fig.  13).  bag  were or  the  spindles  either  mitochondria  surface  the  in  or  either partially  e n c i r c l e d t h e m u s c l e c e l l ( F i g . 13).  Quantitation and The (Table slow  total  I).  No  and  fast  accurate  Distribution number  of  muscle spindles per  dramatic  differences  muscles  of  assessment  of  either  spindle  were the  density  muscle was tabulated  noted  in  mean  normal  or  dystrophic  in  two  muscles,  25  the  spindle  for  numbers  animals. the  e a c h age  group  between For  the  a  more  muscle-spindle  index  F i g u r e 12 L i g h t m i c r o g r a p h of a transverse plastic section o f portion of a 3 - w e e k dystrophic soleus, stained w i t h toluidine blue. T h e p o l a r s p i n d l e i n the center o f the field ( a r r o w ) l i e s c l o s e to a n e u r o v a s c u l a r t r u n k a n d a p p e a r s n o r m a l . A n e i g h b o u r i n g area o f muscle (asterisk) contains a plethora of degenerating a n d a b n o r m a l extrafusal fibers a n d c o n n e c t i v e t i s s u e c e l l i n f i l t r a t i o n . B a r = 2 0 p m ; x 1,100.  26  27  F i g u r e 13 Electron micrograph of a portion of a juxtaequatorial spindle from a 3-week dystrophic soleus. P a 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 s a t e l l i t e c e l l (Sat) a r e indicated. Outer (OC) and inner (IC) capsular components and a sensory nerve terminal (arrow) appear normal. T h e p e r i a x i a l s p a c e ( a s t e r i s k ) is c l e a r . Bar=lpm. xl3,600.  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 of part of an equatorial spindle f r o m a 3 - w e e k dystrophic E D L . Inner capsule cells a n d their processes (IC) e n v e l o p two pairs o f bag (B) a n d c h a i n (C) fibers and their sensory terminals (arrow). A c l e a r p e r i a x i a l s p a c e ( a s t e r i s k ) is surrounded by an intact outer capsule ( O C ) . Bar=10 u m ; x4,000.  28  was  also  total  calculated  number  of  for  each  spindles  age  in  a  Whereas  mean  muscle  weights  animals,  mean  spindle  numbers  group.  This  to  3  would  weeks  parameter  for  all  either  E D L for a given The  from  serial  Prior  to  sides  and  positions  could  be  determined.  made  dystrophic and  EDL  dystrophic absolute Typically,  (Table  of  which  of  I).  No  muscle  spindles  weeks  index  is  defined  as  milligram  of  muscle  weight.  age,  reflecting  remained  in  mean  obvious  dystrophic  muscle.  of  extending were  tendon  spindle  and  growth  constant  for  muscle-spindle  differences  animals  15  or  be  the  pattern  of  age  spindles  in  the  both  origin in  or  entry were  each  were  index  compared  to  the  muscle from  apparent the  the  of  each  between  1  in  this  soleus  and  was  were  pathway.  30  a  way of  estimated  that  the  was  from  (Figure  9-14  and  and  lateral bundle serial  reconstructions  were  both in  a  16).  along  16).  of  in  that  the made  and  number  in  and soleus  in  muscles  of  The  range  in  the the  normal normal  observed  type  concentrated  medial  the  spindles also  15  neurovascular  studied  of  in  were  (Figs.  Longitudinal group  that  reconstructions insertion  point  localization muscle  muscles  to  distribution  soleus  was  such  identified.  depicts  of  in  lengths  from  regardless  spindles  the  soleus  Longitudinal  mounted  muscle  typical  the  from  and  could  soleus  Figure  in  mice.  Muscle spindle  This  animals  dystrophic  muscles the  the  animals.  the  3  per  with  decrease  and  sections  the  EDL  number  1 to  gradual  expressed  steadily  normal  and  transverse  an  from  groups  normal  sectioning,  for  muscle  muscle-spindle  group.  for  in  The  increased  the  between  EDL  sections  given  explain  distribution both  group.  EDL  9-12.  neurovascular  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 a n d d y s t r o p h i c ( D Y ) s o l e u s 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 a g e .  1 WK No. Index  2 WK No. Index  No.  3 WK Index  SOLEUS N DY  9.5 10.4  9.0 8.2  12.0* 10.0  4.2 3.3  11.3 10.4  2.8 2.9  EDL N DY  10.5 10.4  8.0 7.1  11.2 9.2  3.8 2.9  11.3* 9.2  2.5 2.4  (N)  * P < 0 . 0 5 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 - t e s t . C o m p a r i s o n s w e r e also m a d e b e t w e e n the soleus a n d E D L across a l l g r o u p s , b u t these d i d not p r o v e s i g n i f i c a n t at p < 0 . 0 5 . n=5, w i t h the e x c e p t i o n o f the 1-week 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 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 t h e s o l e u s ( 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 a g e . E a c h line i n the diagram designates a spindle. 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 t e r a l ( L ) b o r d e r s of each muscle are indicated. Figure 15b depicts the corresponding transverse reconstructions of the n o r m a l soleus 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 of muscle 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 dot represents a muscle spindle. Dorsal (D) a n d v e n t r a l ( V ) s u r f a c e s a r e i n d i c a t e d as w e l l as t h e m e d i a l ( M ) and lateral (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 r a n s v e r s e s e c t i o n is 1 0 0 0 u m .  32  F i g u r e 16 Longitudinal 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 t h e d i s t r i b u t i o n of muscle spindles in t h e d y s t r o p h i c s o l e u s ( 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 a g e . A s i n F i g u r e 15, e a c h l i n e r e p r e s e n t s a m u s c l e s p i n d l e as t h e y w e r e o b s e r v e d f r o m t h e o r i g i n ( O ) to insertion (I). Figure 16b shows the corresponding distribution in transverse reconstructions. E a c h dot indicates a muscle spindle and intervals between traced transverse sections are 1000 u m . M e d i a l ( M ) , lateral ( L ) , dorsal (D) and ventral borders are i n d i c a t e d .  34  © H \0OfU  ®  SOL  EDL  DISCUSSION  The spindles  results in  process  the  dystrophic  (Meier,  shown  of  that  murine  1969;  neonatal  present  study  complement  muscles  Yellin,  1974b;  spindles  in  are  morphologically  James  the  previous  and  dystrophic  Meek,  observations unaffected  1979).  slow-twitch  In  soleus  that  muscle  the  disease  by  addition,  and  we  have  fast-twitch  EDL  muscles resemble those f r o m their a g e - m a t c h e d n o r m a l controls. Our also  results  indicate  confirm  that,  in  dystrophic  muscles  dystrophic  animals,  contrast  bag ,  bag  x  reactivity  staining  characteristics  rat  after  (Soukup,  previously to  the  differentiate  ATPase  the  those  acid  1976;  to  Banks  chain  and  similar  changes  normally and  2  reported  et  in  al.,  three  fibers  could  in  Khan  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 ,  types. be  In  both  Soukup,  1981), m o n k e y  on  and  1979),  but  fibers  normal the  Regional  muscles  1974b)  intrafusal  differentiated  other and  Yellin,  fibers,  preincubations.  noted  1977;  1969;  extrafusal  into  alkaline those  (Meier,  and  basis  variations  species, cat  of in  namely  (Banks  (Ovalle and Smith,  in  et  al.,  1972),  and  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 also o b s e r v e d i n the m o u s e . We stable  have  and  display  regions  have  high  activity.  spindle, capsular rabbit, alkaline acid  low  alkali  These of  differences results  shown  show  rat  that  t  (1977)  in  In the  mouse  reported Banks are  more  similar variations  to  in  in  the  contrast,  are  are  observed  to  reports,  previous  compared  alkali the  degree  coworkers reactive the  bag  noted  by  36  to  activity  2  alkali  us  in  On bag  x  extracapsular  polar  the  slight  fibers  length  bag  1  the from  fibers  regions  the  our  of  varies  other of  regional  Whereas  polar  the  and alkali  stability  noted  acid-  high  entire  extracapsular fibers.  with  species.  the  are  acid-stable  however,  over of  in  other  (1977)  in  mouse  acid-labile  consistently  that and  fibers  2  fibers  when  low  bag  fibers,  2  chain  contrast  has  regional  Bag  were  exhibit  feature  species,  whereas  fibers  spindles a  other  patterns  regions.  cat  the  in  reactivity,  bag  conditions,  conditions,  activity.  spindles.  polar and  alkali  observed  Kucera to  as  staining  muscle were  that,  hand,  in  under under  mouse  are  similar  to  followed  those  reported  from  the  in  polar  other end  species.  toward  Thus,  the  as  serial  equator,  the  sections  of  staining  a  spindle  pattern  are  gradually  c h a n g e s 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 g h t . It fibers  has  been  may  be  hypothesis, rat  correlation  normal  histochemical that be  regional  by  disputed  of  by at  myosin  Zelena and birth  and  cholinesterase fibers The  innervation  in  Soukup  well,  the  location,  integrity  be of  Kucera  regions,  by  intrafusal  by  that  and  mature  to  (1981)  found  no  type  of  or  the  that  the  same  factors  may  instead  fibers  sensory  This  showed  suggested  governed  or  intrafusal  1973).  number,  He  the  the  who  differentiate  As  not  of  Milburn, (1974)  staining.  may  polar  staining  1969;  eventually  activity  typing.  ATPase  (Yellin,  characteristics.  intrafusal  fiber  beta  by  in  innervation  ATPase  determined  extrafusal  influenced  motor  de-efferented  response  control  variations  histoenzymatic  between  endings  on  has b e e n  selectively  their  motor  dependent  however,  spindles  obtain  suggested that  innervation  in  the  c o n f i n e d e n v i r o n m e n t o f the capsule. It spindle  is  may  Bormioli, that to  contain  are  anti-slow and an  possible  1980;  there  slow of  also  Celio, three  anti-slow  different  different  myosin  1981;  et  myosin  myosin,  entire  that  chain tonic  spindle  of  isoforms.  Kronnie,  types  the  to  myosin. with  intrafusal  fibers  Immunohistochemical  1981,1982)  associated w i t h  fibers  stained  regions  anti-fast  have  recently  intrafusal  myosin,  Future  studies  designed  various  antimyosin  an  individual  reports  (Pierobon  presented  fibers.  and  in  Bag  bag  x  fibers  to  evaluate  antibodies  may  2  to  evidence  fibers  react  both  anti-  serial  sections  reveal  regional  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  extrafusal et  al.,  detect changes by  noteworthy fiber  typing  1983)  rat  transitions  in  other  dystrophy  as t h r e e  postnatal  intrafusal  extrafusal in  muscular  as e a r l y  P e r h a p s the  alterations in  that  fiber  fibers proteins  in  in  weeks  of  age range type  in  response  such  as 37  the age  mouse (Parry  selected i n response  to  to  and  dramatically Parslow,  this  study  the  disease.  denervation  parvalbumin  can  and  or  was too  exercise  the  1981;  peptide  affect Wirtz  early  to  Histochemical are  preceded  pattern  of  sarcoplasmic  reticulum  phenomenon  may  (Green  occur  in  staining  et  al.,  response  myosin  ATPase  methods  type.  Immunohistochemical  1984;  to  are  studies  Muntener  dystrophy not  in  sensitive  with  et  al.,  the  1985).  mouse,  indicators  antisera  to  it  is  of  various  nature  of  spindles  the  environment been  outer  of  of  It  capsule, fibers  results  regenerating  deafferented, intracapsular  the myosin  and  et  which al.,  extent  to  myosin  on  abnormal  patterns  fluid staining  reflect muscle  of  is  loss  spindle  the in of  myosin When  in of  bag  muscle  fibers  trophic  capsule,  have may  and  an  internal  any  studies  influence  of  an  abnormal  (Rogers  activity  spindles  and in  are  viable  selectively  and  is  (Kucera,  only  between  diminishing  and  intrafusal  decreased altered  the  mammalian  intact  interaction  thereby  the  capsule  ATPase  fiber  protective  regenerating  of  that  chains  some  face  the  face  significantly  pattern  the  the  light  recently  in  possible  regulate  capsule  survival in  1982).  space  the  formation  the  innervation  (Rogers,  Only  similar  abnormal  by  to  a  type.  disease  known  function  spindle  depends  are  which  normal  that  of  1980).  sensory  ATPase  the  the  shown  muscle  may  (Dow  maintain  periaxial  abnormality  innervation  in  manifestations  capsules,  grafts  Inadequate  however, of  to  been  muscle  spared  inner  investigate  has  1981).  be  receptors  spindle  following  Carlson,  This  to  the  environment. muscle  and  these  conducted  capacity  may  also  early  heavy chains might reveal a more reliable m a r k e r for alterations i n f i b e r Muscle  While  the 1980).  afferent  the  regulatory  f u n c t i o n o f the capsule.  Spindles in s l o w - t w i t c h versus fast-twitch The function higher  muscle (Voss,  in  spindle 1937;  muscles  population  Cooper,  initiating  density  1960;  fine  muscles is  Barker,  movements,  usually 1974).  determined Generally,  maintaining  by the  posture,  the  spindle or  contractile properties ( B a r k e r a n d C h i n , 1960; Swett a n d E l d r e d , 1960; Y e l l i n , Our  observations  histochemically  from  indicate  those  in  that the  spindles fast-twitch 38  in  the EDL  slow-twitch with  respect  host  soleus to  muscle's  density  exhibiting  is  slow  1969). do  not  myosin  differ ATPase  activity.  In  different  between  complement (Soukup, Samaha  soleus  staining  light  and  two  chain  1981).  neonatal for  bag  Soukup  and  noted  for  was  spindles  animals. fibers  that  rat  fibers  ATPase.  and  the  EDL  This in  significantly  the  soleus  muscles  Guth  observed  present  of  spindles in  b o t h the  number  similar  to  Ontell,  1981)  with  displayed  difference  not  and  reacted  consistently  slight  the  findings  EDL  employed in  the  not  histochemical  soleus  however,  chain  were  Our  in  whereas  myosin  reactivities  of  (1976)  preincubation,  pattern  fiber  distribution  in  others  acid  staining  EDL  by  protocol  after  and  muscles  Kronnie,  intensities  typical  number  reported  et  (1970)  the  these  results  1976,  variable the  addition,  study,  in  the  nor  has  it b e e n r e p o r t e d b y o t h e r s . In all  the  age  others  present  groups for  Slightly  was  the  higher  assist  in  show  a significant  spindle  1977).  postural  the  mean  approximately  averages  Kucera,  of  the  soleus ( Y e l l i n ,  1969;  any  study,  a  difference  is  groups not  a  such  rule,  studied. reliable  for  in  the  explanation  measure  when  is  applied  this  of  the  greater  1974).  to  counts  in  soleus f r o m for  soleus a n d E D L  muscles  density  soleus ( B a r k e r ,  density  An  similar  spindle  as t h e  spindle  is  E D L (Kozeka and  published  general  in  This  and  been  maintenance,  neonatal  density  As  ten.  1974a)  have  number  made the  by  mouse.  rat  in  (Yellin,  muscles  that  Our  results  did  that  of  EDL  observation developing  in  the may  not  be  muscle  in  that since,  o w i n g to g r o w t h , t h e w e i g h t o f t h e m u s c l e has n o t y e t s t a b i l i z e d . As  expected,  neurovascular dystrophy in  both  particular previous  or the  the  trunk age  of  soleus  area reports  of of  distribution  in  both  the  animal.  and the the  the  of  types  of  of  was  distribution  to  the  the  This  spindles  This in  ( 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  followed pattern  heterogeneity  mouse,  noted. of  spindles  muscle.  Owing  EDL  muscle  muscle  no  was  of  course not  extrafusal  segregation  observation  the  of is  selected h i n d l i m b  the  altered fiber  spindles in  of  by types  within  agreement  muscles of  the  a  with rat  Morphogenesis Muscle Ontell,  1981),  definitive sensory  for  the  by  of  4  their  findings  of  Landon  (1972)  formed the  in  Kozeka  by  myotubes  primary  rat,  of  age  are  still  fiber  myotube  of  intrafusal  fibers  is p r e s e n t  an  both  soleus  and  the  noted  at 2  frequently a  fiber  3 weeks of  stained  distance  weeks.  and  that  primitive  It lags  is  possible,  behind  mammalian  bag  1981;  Barker  fibers  share  fibers  and a  that  single  close  1 week  support  1981).  that  age i n  ATPase  staining  as t h e the  the  develop  fiber  2  capsular that  other in  bag  the  compartment  it  has  also  within  a  40  fibers  differentiation  proposed  new  addition,  the  is  polar  hypothesis of  at  reported  sensory  The by  intrafusal the  newly  region  relationship  of  has  histochemical  profile  genotype.  at  same  1  At  week  was  1 week  of  the  age, the  acid conditions  This  fibers.  been  events  1973).  close  adult  was  a  it  has  that  spindle  been  2  at  of  2 the  and  1979,  for  or  3  bag  reported  fibers  (Butler,  that fiber  x  (Kozeka and  bag  In  as  bag  of  F i g . 1)  observed  each other  shown  developing  not  (see  differentiation  While  close association w i t h  1984),  present  (Milburn,  with  histochemical  intrafusal  be  In  mouse, regardless o f  under  zone.  primitive  type.  the  the  to  obtain  In  This  The  Furthermore,  generations cells.  1973).  intrafusal  not  the  and  myotubes.  similar of  postnatally  affiliation  indicate of  do  (Kozeka  days after  1973).  and  successive  Ontell,  study  therefore,  Milburn,  days  age w i t h one e x c e p t i o n .  into  of  state  mononucleated in  (Milburn,  than  complement  o f the i n t r a f u s a l f i b e r  myosin  as i n t e n s e l y  extended  that  rat  utero  has b e e n s h o w n  Milburn,  strongly  15 i n  developing  earlier  full  12  (1981)  (1973)  present  EDL,  until  and  is  1972;  the  and  adults  immature  originate  by  the  day  is e s t a b l i s h e d 2  fibers  this  type  (Kozeka  the  nerve  at  in  mouse  Again,  in  of to  the  where  Ontell  fusion  reported  (Landon,  Milburn  appear  results  that  begins  in  profound influence on differentiation The  mouse  myotubes  1981).  and  and  between  the  adult  occur  the to  intrafusal  Ontell,  fibers  distinct  myotubes  of  days  these  prior  made  spindles  established  of  are  and  days  in  muscle spindles in  number  (Kozeka  regions  3  of  contacts  muscle  development  about  number  addition, birth  spindle  x  that  Ontell, chain 1980).  Thus,  if  expect bag  bag  the  fibers  x  time  in  the  course  mouse  of  their  were  to  develop  histochemical  as  a  separate  differentiation  to  group, vary  one  from  might  that  of  may  be  and chain fibers.  2  The  variability  influenced sensory  by  the  region  been  shown  1957)  and  to  in  gradual the  that  differentiation  that  elongation  exposed  sensory  into  polar  of  ends  innervation  fusimotor  is  innervation  characteristics  (et  Kronnie,  1982),  differentiation  o f f i b e r t y p e is u n k n o w n .  the  for  required of  fiber  capsule  and  necessary  degree  intrafusal  spindle  (Kozeka  is  the  distinct  Ontell,  the  influence  from  the  1981).  spindle  for  types  equatorial  While  it  differentiation development  of  the  (Zelena,  of  spindle  has  myosin  capsule  on  Functional and clinical considerations In  agreement  spindles group  in  the  These and  dy  examined  enlargement  with  of  the  the  observations  Yellin  intrafusal  fiber  innervation  are  the  dy  the  strain  findings  in  1977).  While  some  et  al.,  noted  terminal  stages  of  muscular  James  appeared  or  and  of  light  fiber  dystrophy  in in  none  of  the  at  any  age  signs  of  capsule could  studies  thickening,  be  of  detected.  Meier  (1969)  P a t h o l o g i c a l c h a n g e s s u c h as  of  dystrophia  (1979), abnormal  atrophy  mouse.  thickening  alterations  no  microscopic  dystrophic  human  Meek  ultrastructurally  intrafusal  earlier  collagenous  common  of  Morphologically,  space,  confirm  splitting,  Maynard in  study.  periaxial  in  findings  muscles  present  also  (1974b)  initial  dystrophic  2 J  in  the  the  capsule,  myotonia  spindle humans  and  (Swash and  abnormal Fox,  1975; been  morphology  have  also  (Swash  Fox,  1976)  and  and  m i c e ( D o w and O v a l l e , 1985), their f u n c t i o n a l implications r e m a i n enigmatic. In muscle and  spindles  Walton,  spindles that  human  is  seen  Duchenne are  affected  1968).  In  diminished, in  more  and is  congenital  muscular  related  the  preclinical  and  advanced  the  or  degree  cases.  to  early of  It  stage  of  the  dystrophy,  pathological  would 41  dystrophy,  appear  the  change  the  extent  disease  process  proportion is  therefore  less that  to  of  which (Cazzato affected  prominent muscle  than  spindles  may  be  affected  may  occur  in  within  nonspecific  the  the  progressive  spindle  long-term  course  during  responses  of  more  that  are  the  disease  advanced  not  and  stages  that  of  directly  related  muscle  spindles  any  changes  dystrophy to  the  that  are  probably  primary  etiology  o f the disease itself. The  complex  polyneuronal  extensive  reviews  (Barker,  However,  our  sensory  and  subtle  knowledge  fusimotor  neuronal  do  occur  spindle  in  the  relatively  dystrophy partially  the  noteworthy allelic not  early  (Walton  that  strain  appear  to  of  a  of  fibers.  fibers  mature  exact is  of  It  (Bray the  is  et  that  muscle, provides  that  is  Boyd  and  If  in  muscle  and  spindles  polyneuronal  for  speculate  that  characteristics is  al.,  the  the  reflected muscular  1983),  observed  innervation,  mechanism  dual  in  Swenarchuk, or  1984).  degeneration  et  that  of  the  human  fiber  (Saito  as  of  alteration  extrafusal  such  to  in  subject  Smith,  structural  noted  influence  the  significance  functional  Montgomery  a defense  been  tempting  reflexes  nerve,  of  the  It  this  1981).  1977;  has  functional  tendon  abnormal al.,  1980;  reflected  neurotropic  development  possible  and  not  and  Gardner-Medwin,  morphologically  mouse  are  abnormal  abnormal  al.,  incomplete.  that  dystrophy,  onset  et  pattern  still  activity  in  an  affect  intrafusal in  and  result  the  of  Kennedy  innervation  in  the  1974;  of  changes  in  innervation  is  it  the  1978),  is dy  does  differentiation  unique  spindle  to  2 J  of  intrafusal  during  incipient  stages o f t h e d i s e a s e . Unfortunately not  fully  been  the  development  understood.  studied  in  This  this  With  may  understand  alteration  in  muscle been  fibers  aid  muscular  calcium-binding  protein and  established  the  as a n  be  neuromuscular  inspection. better  the  may  of  of the  more role  of  alternative  to  the  fact  but  muscle such This  myosin 42  that  they  biochemical  parvalbumin to  abnormalities  the  disease  One  parvalbumin. of  due  sensitive  dystrophy.  degree  spindle  in not  only  have and  spindle  only  is  in  fiber  spindles  received  cursory  typing  in  markers  muscle  marker  associated  is  few  normal  immunoreactivity  ATPase  have  dystrophy  immunological  immunological protein  muscular  could with  muscle  (Celio  and  we  and  its  be  the  fast-twitch fibers  has  Heizmann,  1982).  In  British  a previous  Columbia  slow-twitch changes transition 1973;  of  extrafusal The  adult  the  isoforms and  as a n  and  and  Pette,  noted  this  extrafusal  to  myosin  1979;  Klug  fibers thesis fibers  at  in  will and  (Jasch  response  et  al.,  earlier deal  to  1983b;  may  the  43  at  the  altered  Moase,  system  in  Green  1985).  occur  the  et  al.,  of  1984).  of  parvalbumin  of and  addition, than  (Sreter  ATPase  localization  fast  In  earlier  expression of  myosin  University both  electro-stimulation  than  quantification  dystrophic mice.  and  detect  stage with  Group  significantly  sequestering  marker, an  be  mice  and  immunochemical  intrafusal of  of  Muscular Dystrophy  dystrophic  calcium-binding  remainder  intrafusal  of  The  was  the  Heilmann  parvalbumin,  from  parvalbumin  muscles  in  report  et  the al.,  Therefore, dystrophy  in  histochemistry. parvalbumin in  normal  in and  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  parvalbumin  INTRODUCTION  Historical Background of Parvalbumin Deuticke  (1934)  concentrations low  molecular  from on  using  carp  their  muscle.  of  (Pechere  et of  al.,  1969),  it  More  soon  was  high  became et  al., light  concluded  that  Pechere  with  an  referred  been  al.,  the  of  all  chains). these  and  In  contain  the  the  related  Based  coworkers  lower  on  same  as  2 +  in  which  -binding  peptide a  maps  family  discovered  a  as  (Focant  6  and  muscle  contains  4  main  crystallization  proteins  of  based  given  (Pechere  that  spectrum  many  muscle  isotype  salt  similar  parvalbumins,  three-dimensional Ca  low  absorption  as  muscle,  first  constitute (1971)  the  at  observed  vertebrates,  single  Carp  of  proteins  proteins  from a  muscle  ultraviolet  these  1979b).  subject  1972)  to  frog  subsequently  unusual  isolated  vertebrates  in  (1955)  solubility.  have  et  fractions  Henrotte  water  Haiech  myosin  importantly,  (1968)  higher  1971;  (Nockolds  calmodulin,  sedimenting  proteins  (PV)  whereas  PV,  descriptions C,  and  parvalbumin  1965),  isotypes  acidic Pechere  size  Pechere,  slow  ultracentrifugation.  weight,  small  isotypes  described  (Troponin-  and  Capony,  homologous  proteins.  parvalbumins  have  a  high affinity for calcium. Originally  it  amphibia  and  fish,  detectable  in  was  fish  white  muscle  been  found  in  Strehler, 1982a), 1985).  1979), cat  certain red  carp  thought  reptiles  muscle, P V  (Lehky  rabbit et  Parvalbumins  et  (Lehky al., have  parvalbumins  (Focant  1974).  al.,  al.,  1986),  and been  were  Pechere,  Significant  Pechere,  human  (Lehky  identified  et  1974), et in  al.,  white  Although  0.7%  amounts  either  to  1965).  (Strehler  1974;  44  confined  approximately  1974), c h i c k e n  et  not  and  constitutes  (Gosselin-Rey,  turtle  (Stichel  that  of of  the PV  1974;  it  wet  is  of  however,  Heizmann  (Berchtold Berchtold  invertebrate  of  barely  weight  have,  a l . , 1977; rat  muscle  tissues  and  et  al.,  et  al.,  or  in  smooth  muscle, and  it  is  present  only  in  low  amounts  in  cardiac  muscle  (Gosselin-Rey,  1 9 7 4 , B a r o n et a l . , 1 9 7 5 ; L e P e u c h et a l . , 1 9 7 9 ; G e r d a y et a l . , 1 9 7 9 ) .  Evolution The groups,  evolutionary designated  (Goodman  et  al.,  oj-parvalbumin  a  history  of  and  both  1979).  (pH  f$;  groups  than  5.0)  being  proteins  calcium  ions ( G o o d m a n and Pechere,  I  the  troponin-C  thought  deprivation and  the  to  of  have  the  alkali  myosin  2 +  an  PV  from  a  proteins  ancestral  according  less a c i d i c t h a n  evolved  four-domain  chains  properties are  most  to  into  two  gene  of  isoelectric  similar  to  the  Ca  binding  a deletion II.  points,  The  precursor,  domain  main  duplication  p-parvalbumins.  P a r v a l b u m i n arose b y  -binding  light  these  from  separate  1977).  Ca  divides  derived  two  binding  and  are  These  greater  are  parvalbumins  of  2 +  4  domain  Calmodulin,  ancestral  protein  ( r e v i e w b y G e r d a y , 1982).  Biochemistry of Parvalbumin Despite al.,  1978),  the  it  properties.  is  polymorphism remarkable  Parvalbumin  these  a  heat-stable  and  is  (pi)  ranging  molecular  weight  (Mr)  of  al.,  1973).  absence high  The  or  tyrosine  acid and  its  of  9 -  high  amino  presence  number  glutamic  in  from  3.9  13 K D  diffusion  only  one  phenylalanine  (Pechere tryptophan,  et  PV  of  tyrosine  1973).  shows  the  constant  residues  al.,  in  et  proteins  a  and Due  in  their  physicalchemical  carp  in  to  low  6.6  acidic  The  II)  w h i c h the a b s o r b t i o n bands o f p h e n y l a l a n i n e are v i s i b l e .  protein lungfish,  size o f  residue  per  acids  such  a  protein  is  (Pechere  et  high  ratio  ultraviolet  of  molecule, as  by  the  and  aspartic  phenylalanine  absorption  an  and  this  rate  et  with  is c h a r a c t e r i z e d e i t h e r  amino the  the  sedimentation  tryptophan  characteristic  45  Gosselin-Rey  acidic  and  to  1971;  water-soluble  P V (Table or  al.,  are  ( G e r d a y et a l . , 1 9 7 9 ) .  acid composition  of  (Pechere  consistent  point  reflected  parvalbumins  how  isoelectric  also  of  spectrum  a  and to in  TABLE  Amino  II.  Amino  Acid  Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Cysteine  acid  composition  Rat Brain 15.5 2.2 1.0 14.6 5.2 10.6 9.5 0.0 9.4 11.4 5.2 2.4 6.0 9.6 0.0 8.5 0.0 0.0  of  Rat Muscle 16.0 2.0 1.0 14.0 5.0 11.0 8.0 0.0 9.0 11.0 5.0 3.0 6.0 9.0 0.0 8.0 0.0 0.0  parvalbumin  in  the  and  mouse.  Mouse Muscle 16.0 *  1.9 1.0 13.8 4.2 8.1 12.4 0.9 9.2 11.4 6.0 2.1 5.9 9.3 0.0 9.1 0.0 0.0  T h i s table has b e e n e x t r a c t e d f r o m data presented b y H e i z m a n n , 1984. * I n d i c a t e s t h e a m i n o a c i d h i s t i d i n e w h i c h w a s i o d i n a t e d f o r use i n o n page 70.  46  rat  the  RIA  described  Calcium and Magnesium Binding The proteins and C  a and  basic such  polypeptide  as  troponin-C  nonhelical D  and  structural and  N-terminus.  between  Octahedral  helices  E  and  The  AB  domain  is n o t  1982).  structure  formed  by  EF C  helical high  calmodulin.  The  unit.  All  four  affinity  for  Ca  latter  regions but  2 +  resemble the E F hands o f The et  al.,  role  in  muscle.  myoplasmic of  the  Ca  et  hydrophobic for  simple  four  bind  Ca  also  al., core  competition  have  physiological are  sites  F  ,  2 +  .  the  All  et  case o f  of  a  the  2 +  -binding  loop-a-helix  between  high  and  D  P V differs  contain  Ca  helices  affinity  a l . , 1982; W n u k  helices C  which  in  occur  have  Eldik  other  -binding  2 +  loops  sequence of  and  the  -Ca  also  and  domains  PV  have  are  from  the  basic  for  et a l . ,  called  the  that o f  Tn-  structural  T n - C , sites  calcium  loop  AB  3 and  sites  the  same  of  extensively  our  conditions  (1  mM  Mg  be  10"  M  to Mg  binding  (Haiech  presence  been  clarified  P V are o c c u p i e d b y  and  for  and  estimated  Magnesium  the  These  E  Mg  and  a-helix  -binding (Van  of  1981).  in  2 +  bind  studies  concentrations  sites o f  calcium  have  an  PV  calcium-binding  primary  properties  these Under  2 +  binding  Robertson  PV  and  2 +  to  on  a-  4  have  calmodulin  parvalbumin.  calcium-binding  1982),  Ca  The  is  F.  helices  and C D hands, respectively. and  common  calmodulin,  magnesium. The  unit  et  understanding  al.,  8  (Cox  +  induces  elevated  binding  2  et  )  2 +  of  in  and  its  the  the  resting  A  decrease  in  concentrations  Therefore,  in  Ca  Mg  muscle,  majority  conformational  1979a).  vivo.  et  (80-85%) al.,  change the is  PV  (Wnuk  physiological  a l . , 1979; Potter  a  magnesium  sites.  investigated  1977; at  the  affinity  of  indicative  of  is a l w a y s  in  a  m e t a l - b o u n d state. Association  constants  for  method  of  according  to  the  conditions  of  measurement  1979b). range  The of  considered  apparent  values  for  independent  the  of  determination,  (Benzonana  constants magnesium and  binding  for  et  al.,  calcium  is  equivalent  10  4  the  to  with 47  2 +  type  1972; range 10  5  and  Cox from  M" .  respect  of  1  to  2  to  +  PV et  10  The their  to  differ  investigated,  al., 6  PV  1979; 10  two metal  9  slightly and  Haiech  M" , 1  the  et  al.,  whereas  the  binding binding  sites  are  properties  (Cox  et  al.,  however,  1979;  that  Cave  the  et  al.,  properties  1979;  of  the  Haiech two  et  al.,  calcium  1979a).  sites  are  Evidence different  suggests,  (Smith  and  W o l e d g e , 1985).  Functional Aspects Although biological  parvalbumins  function(s)  is  limited.  and  myosin  troponin-C  (Tn-C)  function  parvalbumin  the  of  common  proteins 1980;  may  evolutionary  (Wnuk  et  W a l s h et  al.,  al.,  have  light  it  to  to  PV  Ca  does  not  2 +  is  substitute no  of  PV  with  to  for  the  other  or  their  calmodulin, that  However,  enzymatic  troponin-I  of  suggest  processes.  known  with  significance  reasonable  -activated  has  interact  homology  it  cannot  Parvalbumin  extensively,  the  chains  related  origin,  1980),  studied  Due  be  1982).  been  Ca  2 +  the  despite  -binding  functions  (Cohen,  tropomyosin,  nor  can  i t b e p h o s p h o r y l a t e d ( B l u m et a l . , 1 9 7 7 ) . In that  light  this  of  the  protein  may  glycolysis  (Focant  and  functions  have  now  soluble  protein  muscle  (Eibschutz  the  et  of  muscle  have  more  a  Pechere,  been  found  breakdown  observed  type  specialized  1965;  attributed  to  eukaryote  al.,  1984)  where  to  it  glucose.  PV,  1984).  calmodulin  many  of  function  Heizmann,  in  glycogen  specificity  cells.  is  its  been  muscle  1980).  present  long-term  However,  has  role  in  many  and  of  these  Calmodulin both  metabolic in  suggested  contraction  However,  (Cohen,  It  mediates  in  it  fast  and  changes  skeletal  is  muscle  a  slow  such  as  calcium-  t r a n s p o r t is less w e l l d e f i n e d . The suggested muscle.  role that  of they  Briggs  parvalbumins could  (1975)  be  had  remained  involved  earlier  in  obscure the  suggested  until  Pechere  and  contraction-relaxation  that  PV  is  the  'soluble  coworkers  process  of  (1977) skeletal  relaxing  factor'  et  1977)  of  muscle. Calcium thereby 1976),  free  completely while  PV  can  inactivate  fragmented  SR  take the will  up  calcium  ATPase return  from  activity PV 48  to  of  Tn-C the  the  (Pechere  myofibrils  calcium-free  al.,  (Gerday state  and  (Gerday  and  Gillis, and  Gillis,  1976; B l u m  mechanism (Pechere, muscle  for et  Heizmann the  Tn-C to  of  (Mg  is  of  2 +  and  )  possible  the  Calcium  the  is  the  binding  binding  of  to  of  of Ca  PV  Pechere cell  due  sites  to  2 +  et  the  the  to  the  affinity  Tn-C  dependent  al  and  (1977) to  PV  allow  Ca  on  2 +  Mg  of  are  the  the  of  is  2 +  and  in  the  magnesium of  step,  ( H a i e c h et  concentration  and  similar  well,  by  limiting  SR,  PV  dissociation  rate  by  troponin-C.  As  occupied  contraction  +  from  Ca  PV.  skeletal  modified  kinetics  The  relaxation  with  for  shuttle  fast  later  parvalbumin  is  in  2 +  interacts  -binding.  2  muscle  Ca  binding  molecule  triggers  the  but  of  as t h e  release c a l c i u m  exceeds that  the  which  during of  different  parvalbumin  Tn-C  SR  works  movement  activated  to  on  probably  immediately  of  by  2 +  is  PV  binding  Ca  is  the  by  dissociate  uptake  illustrating  Although  binding  first  and  by  possible  rate  myofibril  muscle  a l . , 1979a).  must  subsequent  scheme  developed  events  state  which  the  not • trapped  T n - C , the  calcium-free  A  When  ion  Parvalbumin, therefore,  between  was  ( H a i e c h et  that  1977).  1977).  (1984).  sequence  al.,  2 +  17)  liberated  This  Ca  al.,  (Fig.  et  2  +  making  al.,  on  Mg  1979a).  temperature  ( O g a w a a n d T a n o k u r a , 1986). The studied the  actual by  computer  cation  binding  et  al.,  in  approximately  still  distribution  1981).  sites  of  ms.  period  (Gillis,  PV  to  1980).  allow  are  Initially  and  will  relaxation  it  between  saturated of  Ca  at  et  and  al., time  the  Tn-C  1982), of  binding  transported  has  been  back  proposed because  to  the  that  the  of  its  binding  is  a Ca  such  much SR  PV  of  biological  relaxing  as  97%  of  (Robertson is  the  during  sites,  that  pulse  2 +  sites o f  muscle relaxation,  be  muscle  PV  the  to  2 +  following  of  the  1980; G i l l i s  transfer  Hence,  fast  (Gillis,  Tn-C  C o n v e r s e l y , the 200  calcium  simulation  parvalbumin-bound  is  of  achieved calcium  the  recovery  significance  effect  is  due  to  of 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 , 1 9 8 3 ; G i l l i s 1 9 8 0 ; G i l l i s et a l . , 1 9 8 5 ) . The the  kinetics  being al.,  preceding suggest  directly  1981;  hypothesis that  involved  Robertson  et  the in  the  al.,  has slow  been off  rapid 1981).  questioned  rate  decay  of of  Secondly, 49  both  on Ca  tension in  a  the  number  2 +  after  and a  event  Mg  of  accounts.  2  prevents  +  single of  a  twitch tetanus,  Firstly, PV  from  (Johnson PV  et  would  F i g u r e 17 Schematic diagram illustrating the p r o p o s e d f u n c t i o n of parvalbumin (PV) in fasttwitch muscle. I n t h e r e l a x e d state t h e c a l c i u m - b i n d i n g s i t e s a r e b o u n d b y m a g n e s i u m ( M g ) a n d c a l c i u m ( C a ) is s t o r e d i n t h e 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 arrival of an action potential, C a is released f r o m the S R a n d t a k e n u p b y the first a v a i l a b l e b i n d i n g sites 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 (mf). B e c a u s e o f t h e h i g h a f f i n i t y o f P V f o r C a , 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 Ca t h e n b i n d s to t h e f r e e s i t e s . P V is t h e n t h o u g h t to t r a n s p o r t C a b a c k to t h e 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 t h e f r e e s i t e s a n d t h e m u s c l e r e l a x e s ( A d o p t e d f r o m H e i z m a n n , 1984). 2 +  2  2 +  2 +  2 +  2 +  50  RELAXED  STATE  CONTRACTION  action potential  PVCa  TRANSITIONAL  57  2  STATE  quickly  become  saturated  thereby  diminish  its  queries  continue  to  with  relaxing  calcium  effect  demand  a  because  (Gerday,  more  of  the  slow  1982; O g a w a a n d  precise  explanation  for  dissociation  Tanokura,  the  role  rate  1986).  of  PV  and These  in  muscle  contraction/relaxation. The specific sites, the  metal-binding  sites  none  of  of  troponin-C which  calcium-sites  concentration (Robertson binding  to  increases  free  bind  calcium  only  the  fast  Ca  in 2 +  enough  content  of  slightly. to  a  to the  response  play  a  Ca  -Mg  Moreover,  to  sites  of  role  in  2 +  constants  muscle  exchange  contraction-relaxation  is  of  and  calcium troponin,  calcium  cycle,  lower  (pCa),  Ca  et  for  however,  the  that  of  class  the  Ca  exists  adequate  Ca  1981).  2 +  2 +  -  -sites  saturated calcium  increases  of  2 +  However,  The  myosin  Ca  transient  adequately  each  with  of  all  contraction. and  troponin  to  that  al.,  -specific  2 +  binding  response  become  occurred  calcium-  time-course  in  of  off  4 of  than  indicate  parvalbumin, and  the  rate  the  (Robertson  calmodulin  of  contains and  myosin  regulation  those  troponin-C,  expressing and  onto  only  of  much  p C a transient  rapid of  those  models  troponin  -sites  to  concentrations  a single  to  association  parvalbumin  calcium  similar  Calmodulin  similar  skeletal  are  1977).  Mathematical  myoplasmic  2 +  The  troponin,  -specific  al.,  are  in  1981).  calmodulin  et  .  2 +  calmodulin  calmodulin,  in  Mg  calmodulin  al.,  of  (Potter  bind  of  of  et  properties  sites,  only  analogous  -specific  sites  of  troponin and calmodulin. Present calcium Ca  2 +  of  /liter  650-670  the to the  the  the is  to  to  2 +  a  sites  on  fast-twitch  myofibrillar  Ca  2 +  at  rest  during  (Ogawa  higher  range,  PV  (Ashley,  PV  /liter  lowered  have  mM  sarcoplasm  Ca  is  regulatory activate  2-0.05  bound  pmol  temperature appear  in  troponin muscle  -binding  (Ogawa a  and  affinity  1983).  and  Tanokura, calcium  (Haiech  when  At  tetanus  for  PV  capacity  et is  al.,  20°C,  is  a  (Robertson  slower the  is l o w e r et  1986).  Since  1979a),  al.,  to  estimated  further  1986). but  quantities  an  Tanokura,  and  present  52  in  330  This  the  binding  association  than  of  the  umol  of  when sites  rate  calcium  would The  all  increases  accentuated  amount  1981).  bind  be  the  on  PV  than  for  required  predicted  calculated  to  gain  by in  calcium of  3  by Ca  PV  ions  2 +  sequestered the  SR  than  by  dissociation  that  the  must  50%  and  leave  (Baylor  relaxing-factor  et  in  parvalbumin  to  et  al.,  following  are  a  1983).  In from  1983),  further  supporting  muscle.  therefore,  A  have  bound  addition,  calcium  al.,  stimulation,  temporarily  remove  fast-twitch  may,  Tn-C  troponin  S R (Baylor  function  by  the  to  the  is  role  in  that  rate  PV  as  functional  for  2  before  the  out  being  at  decreased  of  the  implications  PV  maximum  myofibrils  disturbance  serious  the  indicates  which  by a  more soluble  capacity  of  regulation  of  intracellular calcium and muscle contraction.  Intracellular  Distribution  Parvalbumins has  been  reported  Berchtold such  as  1984).  et  al  the  in  a  not  restricted  nervous  1985;  skin,  Such  are  tissue  Gerfen  testis,  et  al.,  spleen,  diverse  to  tissue  alone.  Recently,  (Gosselin-Rey,  1974:  Celio  1985;  al.,  1986b),  kidney,  distribution  muscle  Endo  bone,  et  teeth,  indicates  that  and PV  the  and and  have  presence  Heizmann, in  ovary  may  their  other  tissues  (Berchtold multiple  1981;  et  al.,  functions  associated w i t h various c a l c i u m - d e p e n d e n t processes. The  distribution  summarized  in  Table  immunological In  addition,  or  with  when  of III  on  response no  the  in  primarily  page that  cross-reactivity  other  mapping  PV,  the  Ca  2 +  Studies  has  of  using  have  cross-reactivity  -binding  distribution  57.  determined  is  immunological  shown  limited  to  been  observed  between  proteins  (Gerday,  1982).  this  protein  by  that  PV  has  closely  different It  immunological  is  methods, a  restricted  related  species.  isotypes  therefore  means,  to  is  of  PV,  important  use  antisera  s p e c i f i c to a p a r t i c u l a r s p e c i e s . Early of  fish  presented as  well  studies  and  of  amphibians  indicating (Lehky  parvalbumins  PV  are  et  (Focant  that al.,  concluded  the  1974;  present  to  that  and  some  localization  Pechere,  distribution Pechere,  its  of  1974).  degree  in 53  1965).  PV  A  included  More all  was  restricted decade  tissues  recently  vertebrates  it  to  later, of  has  including  white  muscle  evidence  higher  was  vertebrates  become  clear  man  (review  that by  W n u k et a l . , 1 9 8 2 ) . Even  though  distribution muscle, with  differ  high  myoglobin in  compared  to  type  2  type  metabolism analysis  typed  as t y p e  type  their  PV  point  1978).  muscle  (Le  Type  content.  fibers)  Peuch  PV  the  In  fibers  2A  electric  the et  are  al.,  there  spot No  with are  a  and  other  Therefore,  is  et  spot  found in  fiber  could  be  exceptions  hand,  the  (with  is  relationship  and  by  PV  when Within of  fibers  molecular on  weight the  gels  properties  this  vary  categorization.  predominantly  absent  PV  anaerobic  those  identified  and  is  electrophoretic  Only  to  1976)  PV  type, fibers  aerobic  characteristic  Siegel,  muscles  is a c o n t i n u u m  1982).  muscles  white  H e i z m a n n , 1982).  supported  and  in  red  extrafusal  the  al.,  the  few  eel (Childers  1979).  1  quantity  fast-oxidative-glycolytic  red  the  to  following  at  in  on  regard  2 B , a n d 2 C ) there  gels.  fibers  noted  type  finding  are  are  (Celio and  closely  PV  present  chicken,  With  (Heizmann  a large  However, of  of  1982),  two-dimensional  fibers.  2A,  their  contents  levels  in  staining  proteins,  PV  low  absent  histochemical  muscle  produced  on  amounts  oxidative al.,  single  This  High  a l . , 1980).  (types  Heizmann,  cells.  2B/FG  1/SO  Significant  et  these  isoelectric  from in  in  and  et  ATPase  fibers  muscle  while  virtually  myofibrillar  (Celio  type.  1977)  (Hamoir but  of  essentially  muscle  Gerday,  fibers  categorization  of  judged  2  are  to  contents  standard  concentrations  and  according  (Gosselin-Rey and  abundant  the  parvalbumins  slow-  carp (Gosselin-Rey from  between  high  white PV  breast  and  low  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 . , 1 9 7 9 ) d o e s n o t h o l d f o r a l l s p e c i e s . More and  the  react the  is  the  correlation  of  development  of  the  SR  and  strongest  toward  the most  convincing  well  developed  fibers  (Celio with  PV  the  S R , s u c h as t h a t  in  immunoreactivity  Heizmann, fastest type  1982).  relaxation 2/FG  with  relaxation  Parvalbumin times  fibers.  A  and  time  antisera  consequently  continuum  of  PV  s t a i n i n g i n t e n s i t i e s r e s u l t s as t h e s e 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 t h e m u s c l e . In fibers workers  muscle but  in  spindles, P V none  suggested  of that  the the  has  been  nuclear  localized in  bag  intrafusal  one  of  the  fibers  (Celio  and  chain  fiber  probably  54  nuclear  Heizmann, has  chain 1982). faster  intrafusal These relaxing  properties  than  study  the  of  do  the  bag  regional  fibers  or  distribution  of  the  non-reactive  PV  in  chain  intrafusal  fiber.  fibers  A  does  comprehensive  not  exist  at  this  time. Of  the  non-muscle  contain  significant  muscle  tissues  PV,  the  only  tissues  quantities  cross-reacts non-muscle  of  examined, P V (Table  with PV  muscle  that  the  cerebellum  III).  The  anti-PV  has  been  PV  neocortex  found  serum  fully  and  in  many  (Heizmann,  isolated  and  of of  the  brain  these  non-  Rat  brain  1984).  characterized  (Berchtold  et a l . , 1 9 8 2 b , 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 . Parvalbumin cytosol. 1977)  appears  Indirect has  localization diffuse  a  any  particular  uniform  particular  completely  morphological  be  a  soluble  protein,  freely  applied  to  muscle  immunofluorescence  shown to  to  out  and  intracellular  of  the  biochemical  structure(s)  distribution  within  cells  of  PV  organelle.  (Gillis  studies the  carp  et  indicate  cell  even  in In  myofibrils  though  The  is its  not  et  with  muscle  1979). PV  throughout  (Benzonana  skinned  al., that  dispersed  al.,  no  1975,  specific  fibers,  data  PV  will  from  associated  relationship  the  these  with  with  any Ca  2 +  -  r e g u l a t i o n suggests that it m i g h t be. Ultrastructural and  Celio,  1986).  (Zuschratter associated  et  localization  PV  in  cells  P r e - e m b e d d i n g staining al.,  with  1985)  and  microtubules,  Post-embedding  techniques  concluded  that  PV  the  Similar  cell.  of  was kinds  rat  of  intracellular on  rat  have  not  and  brain  is  u s e d to  inconclusive  study  Celio,  membranes  PV  1986),  and  in  (Heizmann zebra  revealed  postsynaptic  finch  labelling densities.  cerebellum  (Heizmann  and  Celio,  dispersed  throughout  the  cytoplasm  homogeneously  studies  the  techniques  (Heizmann  performed  indeed  of  been  published  on  muscle  or  1986)  other  of  non-  muscle tissue.  Quantitation of Parvalbumin Parvalbumin been  localized  has in  been a  identified  wide  variety  in  every  of 55  cells  mammalian by  species  studied,  immunohistochemical  and  it  has  techniques  (Heizmann, cells  or  amount its  between of  PV  biological  and  their  somewhat high  tissue  types  contained  to  the  of  tissue  1983).  is  various  III  of  a  the  and  of  the  is  percent  efficient  PV  of  (HPLC)  PV  examined  commonly  peptide  also  the  with  date vary  methods, for  this  of  to  may  used  analysis  been  of  understanding  conventional  obtained  for  has  the  tissues  more  individual  Estimations  concentrations  now  yield  tool  in  various  of  Instead  quantification  procedures (Berchtold  the  between  tissues.  t i s s u e s has a i d e d  Reports  high  considerably  particular  summarizes  detection.  sensitive  differs  within  content.  of  Immunochemical  developed R I A  as  chromatography  because  HPLC  content  well these  PV  method  liquid  its  Table  corresponding due  as  within  significance.  addition,  al.,  Quantitatively,  performance  quantities In  1984).  small  technique.  (Berchtold  successful by  et  recently  et a l . , 1 9 8 5 ; E n d o et a l . , 1 9 8 5 a , 1 9 8 5 b ) a n d  ELISA  methods ( L e b e r e r a n d Pette, 1986a, 1986b). The  aim  concentration  of  and  fast-twitch  this  section  distribution  EDL  and  the  of  of  the  PV  in  slow-twitch  intrafusal soleus  to  document  and  extrafusal  muscles  dystrophy  is  progressive  regulated,  this  study  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  weeks  age  were  chosen  signs  dystrophy  and  by  mouse light types,  32-weeks PV  was  dystrophy,  since a  age  raised i n  microscopic and  of  level.  because  the  Since  correlation  and PV  in  between  PV  clinical  d i s e a s e has  rabbits  alterations  and  reached an  used for has  fiber PV  been types and  appears  shown are fiber  to a  be  type  to  be  have  was  in  the  of  the  genetically muscular  developmentally  age.  localized  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  and  A n i m a l s at  not  yet  An  localization  common  fibers  Because  a d v a n c e d state.  immunological  changes  muscle  techniques.  content  of  the  normal  by  disease  immunochemical  of  mice  of  and  was  dystrophic  a  biochemical  thesis  in  feature  appeared  antiserum of  PV  specific of  conducted  2-  at  to the  fiber  muscular using  a  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 content i n various species a n d tissue.  Species / T i s s u e Mice gastrocnemius soleus EDL Tibialis Anterior gastrocnemius Tibialis Anterior quadriceps Dystrophic Mice gastrocnemius Tibialis Anterior quadriceps  Reference  PV  H e i z m a n n et a l . , 1 9 8 2 (HPLC)  4.9 g / k g w e t 0.01 4.4  K l u g et a l . , 1 9 8 5 (electrophoresis)  4.8 5.53 m g / g w e t 4.86 5.82  Content  wt  wt  K l u g et a l . , 1985  3.24 m g / g w e t 2.98 3.44  P f y f f e r et a l . , 1984  5 % total heat stable p r o t e i n  gastrocnemius soleus EDL Tibialis Anterior b r a i n (total)  H e i z m a n n et a l . , 1 9 8 2  3.3 g / k g w e t 0.004  cerebellum cerebrum  B e r c h t o l d et a l . , 1 9 8 4 (HPLC)  15 m g / k g w e t 2  cerebellum cerebrum retina lung,liver,spleen  E n d o et a l 1 9 8 6 b (RIA)  prostate bone testis skin  B e r c h t o l d et a l . , 1984 (HPLC)  3217 n g / m g T S P 128 1710 10 24 m g / k g wet wt 15 11 86  brain  cultures  wt  Rat  S c h n e e b e r g e r et a l . , 1985 (HPLC & RIA)  2.4 2.7 0.02 g / k g wet  Horse H e i z m a n n et a l . , 1982 deep gluteus  0.001 g / k g  Man vastus, triceps  0.001  Guinea Pig soleus sartorius  0.007  Rabbit EDL soleus vastus  wet  0.25  L e b e r e r & Pette, 1986a (ELISA) (superficialis)  544 p g / g 4.4 1200  57  wet  wt  wt  wt  MATERIAL A N D  METHODS  General Normal mice  of  (+/+)  the  breeding  used  as  the  of  and  in dy  were  of  /dy  2 J  the  /dy  adult  male  obtained  from  our  Jackson  system dy  normal  2 J  /dy  were  were  system  to  this  mouse  was  Bar  used  pairs  or  as  +/+  M E  dystrophics dy  were  intercrossed  2 J  all  were  /dy  and  mice  with  system  female Original  Harbour,  successful for  breeding  male  colonies.  Sibling  backcrossed  Heterozygote  This  modifications  own  neonatal  1981).  animals  2 J  and  Laboratories,  (Flaherty,  mice  progeny.  2 J  2 J  the  heterozygotes.  2 J  Some  )  2 J  from  homozygous  +/dy  project.  (dy  cross-intercross  Some  resulting  strain  purchased  the  controls  production in  were  by  experiments. mice  dystrophic  C57BL/6J  pairs  maintained  and  were for  all  dystrophic  2 J  adult  for  the  mice  necessary  used  for  the  of  this  neonatal age g r o u p . Clinical strain  signs of  until  intercross only  3-4  muscular  weeks  offspring  homozygote  is  (Atwood  matings  difficulty  mounting  offspring  (Younger  and  dy  dystrophic  animals,  1960;  2 J  Tanioka  techniques.  strain their  et  In  is  al.  not  mates,  addition,  for  whereas  manifested  1978) less  mice  prolific  Silverman,  1973)  not  animals  used a  are Kwan,  for  were  murine  /dy  and  unreliable  dystrophic  2 J  dystrophy  breeder.  1984).  3  1  Males often  ovarian  and  artificial  insemination were  2  use  are  of  often  of  heterozygote Therefore,  of  age.  infertile  problems  (Wolfe  (Jones and  to  This  and  have  delivering  undertaken  transplantation  performed  mice  age.  weeks  were  included  amputations  the  have  Methods  young  weeks  and  and  hindlimb  the  that  than of  females  so  in  to  obtain  and  Krohn,  Southard,  identify  their  mice  1962) of  the  a p p r o p r i a t e g e n o t y p e i n t h e 2 - w e e k age g r o u p . Two-week-old (+/dy ^) 2  females each  and (dy  2 J  mice  normal /dy  2 J  2-week-old  ).  were  obtained  female  mice  Animals  were  heterozygote  from  carrying  a  ovaries  anesthetized  (+/dy ) 2 J  mouse 58  cross  with  was  between  heterozygote  transplanted ether,  amputated  and  from the  proximal  males  homozygote  right  leg  from  to  the  knee  joint.  The  according  EDL  to  analysis.  the  At  tied,  the  heat  lamp  checked  the  skin  the  to  immediately  rungs top  was  of  the  was  survived  surgery, and  cage  balance  top  with  and  14 o f  general  biochemical for  each  outline  for  aspects o f  this  experimental  characterized gels  from  paper a  and  in  both  which  age  the  Department  of  proteins with  (RIA)  to  dystrophic  left  is  to  involved  depicted unless  antisera. in  Dr.  was  modification  or  dystrophic.  in  in  otherwise  mice  a  were  according  to  inability grasp  while  the  31  From  the cage  animals  this  group,  of  the  immunocytochemical 18.  of  Brooke  to  content  British  typing  sulfate  transferred  Baimbridges's  by  size  was  (SDS)  assessed  laboratory  standard  myofibrillar  and  Kaiser  EDL  and  and (n) was slab  nitrocellulose  in  Columbia.  the  for  sample  Parvalbumin  dodecyl  Parvalbumin K.  The  specified.  sodium  accomplished fiber  the  Figure  electrophoretically  University PV  be  under  foot,  inability  were  experiments.  one-dimensional  PV  of  and  and  amputations,  judged  tail,  leg  34  were  the  hind  total  Histochemical  a  considered  of  were  of  age, the  Out  The  localization  recover  rungs  mice,  on  to  the  developed  methodology.  according  5  vessels  climbing  work  the  Physiology,  immunohistochemical antiperoxidase  was  groups  immunoblotted  radioimmunoassay  conducted  group  blood  while a  by  histochemical  foot  methodology  thesis  the  allowed  processed  and  3 weeks of  were  of  major  and  spun  these  the  At  Animals  the  were  mothers.  10 m i c e w e r e u s e d f o r t h e i m m u n o h i s t o c h e m i s t r y A  out  being  left  down.  of  mice  clenching  after  the  upside  surgery  and  endings  and  their  dissected  immunohistochemical  free  dystrophy.  splaying  recover  the  to  subsequently  for  clamped,  them  signs o f  tests:  were  described  sutured  suspended the  of  returning  following  muscles  methods  clinical  to  soleus  time  before  for  and  by the The  peroxidaseATPase  (1970)  was  method  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 . , 1 9 8 3 ) .  Characterization of Parvalbumin Parvalbumin 32-week  and  was  initially  2-week-old  characterized  mice  by  the  in  samples  two-dimensional 59  of  gel  soleus  muscles  of  electrophoresis  method  of  F i g u r e 18 Schematic illustration outlining the general m e t h o d o l o g y followed i n this p o r t i o n of 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 w a s i s o l a t e d o n isoelectric f o c u s i n g ( I E F ) tube gels. T h e b a n d was excised and e m u l s i f i e d i n P B S and Freund's complete adjuvant, a n d this solution was i n j e c t e d into rabbits. The a n t i s e r a (1 A B ) c o l l e c t e d w a s t e s t e d 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 immunocytochemistry (ICC), radioimmunoassays ( R I A ) and immunoblots. B. T h e actual 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 extensor d i g i t o r u m longus ( E D L ) a n d soleus ( S O L ) f r o m both normal and dystrophic adult and neonatal mice. T h e muscles were either 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 t h e s o l u b l e p r o t e i n s u b j e c t e d to o n e ( 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 , or, 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 half and immediately frozen. One half was processed for ICC and the other for myofibrillar A T P a s e histochemistry.  60  METHODS  IEF  EDL and SOL REMOVED  2-D ELECTROPHORESIS  1  RIA  IMMUNOBLOT  61  O'Farrell was  (1975).  identified  work  was  The  in  a series  reported  therefore,  the  protein  left  at  the  hindlimbs.  Teflon  pestle  pH  placed  and  isoelectric  electrophoresis. processed  of  of  ratio  with 1982  and  data  in  authentic  of  of  the  rat  Jasch  32-week  samples  parvalbumin.  and  Moase  the  32-week  mice  were  of  for gels  This  (1985)  samples  and  5 min 5  These were  in  and  was  of  not  subsequently 11  mg  of  subsequently  run  on  5 x  muscle  for wet  the  IEF  lysis  on  for  125 gels  buffer  a  and glass-  tris  buffer  weight  frozen 3  in  x  was liquid  125  mm  two-dimensional  muscle  tube  weight/ml  run  used  with  wet  Mills),  were  cervical right  1 mM  muscle  (Chicopee  samples  of  by  from  separately  containing  used  toto  2 ml  mg  killed  in  homogenized  Miracloth  hr. that  sample mg  and  containing  with  20  aliquots,  135  dissected out  Instruments) for  homogenate  the  dystrophic  weighed  covered  tube  5 mg  concentration  al.,  and  homogenate  tubes  (IEF)  the  weight/volume  were  (TRI-R  Aliquots  with  et  parvalbumin  soleus muscles w e r e  lyophilized  focusing  Jasch  Normal  and  aliquot  then  be  studies  electrophoresis  muscles  polypropylene  nitrogen,  The  EDL  The  An  to  study.  homogenizer  7.4. in  by  Preparation.  and  thought  comigration  two-dimensional  Tissue  dislocation,  of  previously  repeated i n the present i)  band  wet  weight  mm  IEF  was  based  were  tube  described  gels.  on  the  previously  ( O ' F a r r e l l , 1 9 7 5 ; J a s c h et a l . , 1 9 8 2 ) . ii)  Two-Dimensional  week-old pH 5  5-7 x  mice  were  ampholines  125  mm  IEF  Electrophoresis.  partially  (LKB). tube  The  gels.  O'Farrell  (1975),  separating  isoelectric  points.  The  800 V .  The  through  a  culture 2.3%  3 x  30  tubes  ml  125 m m  mM  Tris)  first  were  These  10 at  the  lysis  dimension  parvalbumin  ml pH  from  focused  gels w e r e  syringe.  in  sample was applied  The  samples  containing  S D S , 62.5  solubilized  Lyophilized  removed gels  were  S D S sample 6.8  until 62  for  muscle  buffer  the 17  stored  other  muscle 400  V  glass t u b e s at  -70°C  (10% of  either  according  at  the  from  O'Farrell  run  hr  f r o m the  running  of  to t h e t o p o f was  buffer  samples  in  glycerol,  (1975)  3 x  to  the  by  then  5%  using or  method  of  for  expulsion  16x150  2-  125 m m  proteins  and  the  mm  by  their  1 hr with  at air  capped  mercaptoethanol,  second dimension.  The  5  x  125  mm  gels  125  mm  gels, but  2  hr  at  water, then  room 17.25  gm  tube  gels  Kodak  Blue  a  tubes  were  in  3  solution and  were  days  photographed  in  57.5  gm  and  on  then  the a  the  7 mm  same m a n n e r  diameter 150  ml  methanol,  trichloracetic  gm/50  rinsed  dark,  with  with  fluorescent  ml  light  destain  to  two  box  350  acid.  destain  one  as w i t h  the  3  glass tubes a n d f i x e d ml  The  8% acetic acid before  ( E a s t m a n , 0.0287 gels  in  containing  2 5 % ethanol  The  for  glass t u b e s  placed into  acid  R250  bath.  solution  distilled  gels  for  and  solution  with  for  were  being stained  solution)  with  30  min  stored  changes.  Technical  x  in The  Pan  2415  film.  Comigration samples and  in  30 m i n  the  then  sulfosalicylic  water  culture  from  were  temperature  Brilliant  a 60°C  capped  removed they  destained for  Coomassie in  were  on  IEF  Moase,  soleus  of  authentic  and  muscle  two-dimensional  1985).  homogenate  rat  Comigration on  5  x  125  of  mm  parvalbumin  gels  was  pure  rat  IEF  conducted  muscle  gels  with  was  in  the a  in  homogenate  previous  parvalbumin  performed  EDL  with  one  study the  (Jasch  dystrophic  experiment  in  the  p r e s e n 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 ( 1 9 8 5 ) . The  second  dimension  S D S s l a b g e l s , 17 c m x the  top  of  the  acidic  end of  notch  was  tube  made  kilodaltons  in  (KD);  serum  to  the  described  by  Once  dye  the  (66  the  deampholizing  I,  end KD)  front  solution  the  of  The 3 x  2%  agarose,  for  molecular  10  KD;  exponential  125 m m in  down  gel.  for  migrated for  All  obtained  (1975)  30 m A  5-22.5%  migrate  agarose  was  had  on  the  right-hand weight  human  serum  the  were  from  10-12  hr  through  remainder  at  the of  an  stacking the  buffer,  the  slab gel.  A  (B-galactosidase,  13  side of  albumin,  run.  such  the  66  KD;  pyruvate  2 1 . 5 K D ; l y s o z y m e , 14.3 Millipore  constant  gel, about  whereas carried  current 2  hr,  T h e slab gels were  of  the  KD)  bovine out 25  as mA.  amperage  soaked in  5 0 % e t h a n o l , 7% acetic a c i d a n d 0.005% C o o m a s s i e B l u e 63  to  that  Electrophoresis was initial  gradient  gel was adhered  markers  purchased from  Sigma.  acrylamide  I E F tube  S D S sample  43 K D ; soybean t r y p s i n i n h i b i t o r ,  of  O'Farrell  w a s i n c r e a s e d to  with  phosphorylase-a,  acidic  albumin  gel  1 mm.  gel c o u l d  k i n a s e , 57 K D ; D N A s e next  performed  10 c m x  S D S slab  the  was  a  R250  for  36  hr.  After  Coomassie 30  min  were  Blue  in  the  R250,  50%  destained  gels the  were  protein  trichloroacetic for  rehydrated and  acid  several days  2  polypeptide  (TCA)  in  for  7%  and  acetic  hr  in  7%  spots w e r e  0.1%  acid,  acetic  acid  visualized by  Coomassie Blue  vacuum  and  sealed in  0.005%  staining  for  R250.  The  gels  plastic  bags,  and  photographed o n a light 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 .  Antisera production Prior serum  was  control in  2  Rat  to  the  initial  collected  rabbits  purchased crude  injections,  and  mouse  the by  Parvalbumin  samples  were  phosphate The  carefully  buffer  emulsion  rabbit,  7.4  Mouse  obtained  parvalbumin  right  on  estimated on a  from  in  and  and  1 ml  muscle  by  IEF  Gilford  as  a  gels,  pi  and  with  each  standard  spectrophotometer  running for  50  5.45.  into  (Leeds  and  in  normal  1  ml  of  (IEF) these  0.15  M  ( D I F C O Laboratories). the  hindlimb  of  of  Each  parvalbumin  quantities  Northrup)  of  from  focusing  injection.  spectrophotometric  was  resolved  number  emulsified  amount pg  A  (1976).  (HPLC)  was  as  raised  Hubbard  isoelectric  adjuvant  booster The  =  later  was  gastrocnemius  mm  intramuscularily  parvalbumin.  simultaneously  gels  stained  use  chromatography  125  preimmune  parvalbumin  medial  b a n d at  of  for  Lazarides and  Freund's complete  sides  pg  x  ml  parvalbumin  the  mm  50 stored  muscle  liquid  from 5  injected  left  150-200  the  were  described by  performance  resolved  subsequently  approximately  was  scanned  pH  was  alternating  contained band  at  excised  aliquots skeletal  g e l s , as d e s c r i b e d a b o v e , as a s i n g l e p r o m i n e n t bands  approximately  ul rat  procedure high  was  100 and  CalBiochem-Behring.  homogenate  mice.  to  based on purified  from  muscle  rabbits  Antisera  parvalbumin  adult  from  antiserum.  adult  immunization  of  the  readings. before  the  each  injection in  each  pure  rat  Gels  were  bands  were  r e m o v e d , a n d t h e a b s o r b a n c e w a s c o m p a r e d to t h e r a t p a r v a l b u m i n s t a n d a r d . Immunizations days  after  volume  of  the  were  eighth  phosphate  in  week. buffered  2-week  intervals.  The  Approximately  1 ml  saline (PBS),  5 mM  64  of  first blood  bleeding was  was  performed  collected into  10  an  equal  ethyleneglycol-bis-B-aminoethyl  ether  (EGTA) 2500  and  x  0.02%  g, and  sodium  aliquots  azide ( p H  of  the  serum  7.3).  The  blood  was  centrifuged  were  stored  at  mouse  parvalbumin  -70°C.  for  15  Specificity of  min  the  at  antisera  was tested 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 . The total rat  rabbit  of  3  in  times  over  parvalbumin  resulted  in to  a  beaker  for  was  a  the 52  the  30 m i n  at  antisera  -week  raised was  approximately  incision glass  which  marginal  30  vein  containing 5000 x  period,  bled  ml of  to  a total o f  of  the  right  PBS, E G T A  The  ear, and  and  rabbit  5 times  antiserum.  the  g a n d the  whereas  over  raised  which  78  blood  azide.  s e r u m was p o o l e d .  in  were was  The  was  the  weeks.  rabbits  the  sodium  was  antisera  to  a  to  was of  bleeding  by  allowed  Separate aliquots  a  Each  bled  blood  bled  small  drip  into  centrif uged  20  ml,  1.5  ml  a n d 100 u l w e r e s t o r e d 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 The  antibody  methods.  mouse  Triplicate  determine muscle  to  the  titre  PV.  skeletal  enzyme-linked of  the  muscle  of  was  immunosorbent  antiserum  Monospecificity  PV  and  the  its  characterized  assays  (ELISA)  cross-reactivity  antiserum  was  with  evaluated  by  the  were  conducted  purified  by  following  rat  to  skeletal  polyacrylamide  gel  electrophoresis ( P A G E ) and Western blotting, and radioimmunoassay ( R I A ) . i) this et  E n z y m e - L i n k e d Immunosorbant  study al.,  1978;  coated  with  ng/well plates mouse an  was  based o n Kelly  1979).  rat  parvalbumin  dissolved  in  carbonate  washed  parvalbumin  incubation  (SARIg)  al.,  described  pure  were  second  et  that  for  antibody, was  three was  90  min  sheep  applied  at  at  added  anti-rabbit a  (pH  3912  9.6),  doubling  temperature  and  65  the  with  plates  of  to  1:25  1971;  be  to  at tested  1:3200.  a final  90  to  min  alkaline incubation  in  Butler  (Fisher)  subsequent w a s h i n g , the conjugated  used  were  2 5 , 5 0 , 75 a n d  overnight  serum of  test  Perlmann,  assay  incubated  dilutions  After  and  concentrations  immunoglobulin  1:1000 d i l u t i o n .  standard E L I S A  microtest  and  PBS-Tween, in  The  (Engvall  ( C a l B i o c h e m ) at  with  room  (ELISA).  previously  Falcon  buffer  times then  Assay  100  4°C.  The  for  anti-  Following developing phosphatase and  buffer  wash, was  p-nitrophenyl added  measured test  for  was  each  and  the  reaction  absorbance  run  in  The  parvalbumin  and  in  the  (Fig. the  (Sigma, 5 m g / 5  allowed  405nm  results was  was  at  triplicate,  dilution.  mouse  phosphate disodium  on  to  a  continue  Dynatech  preimmune 19)  of  for  1:200  that -  10% diethanolamine 30  M R  rabbit  indicated  range  ml  min.  580  serum the  reaction  microelisa was  titer  1:1600.  The  was  reader.  Each  u s e d as a c o n t r o l  of  A  buffer),  the  antiserum  working  dilution  for  against of  1:400  was chosen f o r all i m m u n o h i s t o c h e m i s t r y experiments. ii)  Tissue  that  were  Preparation used  for  radioimmunoassays razor  blade. in  Immunochemical  one-dimensional  w a s as f o l l o w s .  Two  homogenized  for  20  to  times  four the  2-week which  muscles was  scarcity  500  ul  give  until  and the  at  All  supernatant  electrophoresis  to  in  and  S D S slab  an  times  dystrophic  the  wet  5 0 % the c o n c e n t r a t i o n  tissue  and  of  (31.25  mM  was  into  100  divided  12-18  were  hr.  run.  ul  The  of  5  at  39,000  into  x  g  Muscle aliquots  freeze-dried  supernatant  mM  samples  used  for  45  0.5  were  the  10  mg  Due  to  the were  homogenized  extracts in  normal  of  EDTA  for  and  muscles  was  and  min  in  0.01%  at  used ml  4°C,  for  gel  Eppendorf  stored  RIA  a  EDTA,  The  weight  dystrophic  T r i s , 0.5  with  1 mM  buffer.  which  and  combined  Tris,  wet  Tris  mg  aliquots.  divided  These  final  extracts  minced  were  mM  2-week  weight  centrifuged  were  (62.5  the  were  gels  buffer  volume  homogenates  for  muscles  of  immunoblots,  weighed  adult  approximate  neonates,  final  immunoblotting  lyophilized  muscles were  cold Tris  give  20  an approximate  buffer  azide).  the  vials,  homogenized  electrophoresis,  minced  of  Preparation  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 .  combined  homozygous  to  Tris  sodium and  also  of  combined  were  the  volume  a n d 0 . 0 2 % s o d i u m a z i d e ) at p H  gel  Individual  of  Studies.  was  at  -20°C  stored  in  according  to  1 0 x 1 2 c m s i l i c o n i z e d g l a s s t u b e s at - 2 0 ° C u n t i l t h e a s s a y w a s p e r f o r m e d . iii) the  Immunoblots.  procedure  aliquots buffer  of  the  of  Electrophoretic  Towbin  et  blotting  a l . , (1979).  muscles originally  as d e s c r i b e d p r e v i o u s l y .  The  homogenized  The  of  proteins  samples used in  twenty  lOOpl f r e e z e - d r i e d 66  was in  times  performed these the  experiments  volume  tissue extracts  from  tris  were sample  normal  and  F i g u r e 19 B i n d i n g response o f t h e 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 decreasing concentrations of t h e rat muscle p a r v a l b u m i n (antigen c o n c e n t r a t i o n ) , as detected by an enzyme-linked immunosorbant assay (ELISA). Absorbance was m e a s u r e d at 4 0 5 n m . A n t i g e n concentrations were measured in n g / l O O u l . T h e values given are the means of triplicate assays, normalized at each dilution 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 (absorbance o f 0.065) d e t e r m i n e d b y the preimmune control serum.  67  dystrophic glycerol,  mice  2%  duplicate the  second 25  pg  was  notched  (25  mM  was  at  V.  NaCl, the  of  the  run  lower  as  a  Bio-Rad  cooled  for  hr  nitrocellulose  equilibrated  transferred and  at  high  carefully  for  any  incubated  anti-mouse  5  in  min  horseradish membrane  washes  was  in  H 0 2  was  2  and  was  gels w e r e  All  temperature.  pm  pore  nitrocellulose  (150 dried  V)  mM  1% N G S p H as  membrane  was  at  (1:3000,  photographed  20-30  the  blots  while  wet  in  (Sigma)  washes min  room  and then for  4-chloro-l-napthol  after  incubated  B i o - R a d ) at  in T T B S  and  was  by  with  were  a  in  temperature  a final  100  ml  5 min TBS  which  was first  done  with  repeated  washing  Tris,  500  7 . 5 , to  T e c h n i c a l P a n 2415 K o d a k  then  2  IgG-  hr.  The Colour  containing 20  agitation.  film.  in  Following  dissolved in  distilled  and  and  in T B S .  were stored and protected f r o m light, on filter paper covered w i t h plastic wrap. 69  saturate  anti-rabbit for  mM  washed  min  solution  gentle in  5  room  above,  4°C.  goat  overnight at  further  overnight  The  min  described  (1:200)  membrane  cooled  30  20  buffer  250/2.5).  or  for  of  destained  The  (Model  antiserum  incubations  terminated  protein.  room  0.45  transfer  half  at  solution  and  was  min  parvalbumin  soaking  to  30  for  by  gel  the  of  Bio-Rad)  the  a  ml  (TTBS,  TTBS,  mg  stained  of  For  corresponding  Tween-20  achieved 60  blocking  half  0.05%  was again washed twice  methanol.  reaction  containing  peroxidase conjugate  development 0.015%  remaining  to  500  intensity  serum ( F C S , CalBiochem-Behring),. and was  according  above.  The  supply  calf  gel  in  in  a  3% fetal  The  a  to  power  membrane in  for  washed  binding-sites.  1 hr  above.  was  free  for  described  methanol)  Cell  3  applied  half, and one half  v/v  Trans-Blot  described  10%  in  corners and 20%  run  of  slab gel was d i v i d e d  destained  was  subsequently  gel, and  technique  pi  the  electrophoretically  saline  the  S D S slab  50  on  then  the  gradient  were  with  run  were  After  They  muscle parvalbumin the  reconstituted  (DTT).  rat  glycine,  either  were  two-dimensional  m M  Tris-buffered  cold  exponential  the  the  age,  dithiothreitol  5-22.5% of  of  it  examined  two  weeks  M  end of  one  run  temperature,  2  subsequently  192  on  transfer  the  in  proteins  40  0.1  authentic  and  Tris,  membrane  a  of  At  stained  The  to  and  dimension  blotted.  then  32  SDS, and  halves  control, be  at  water, The  ml The and  blots  The spots from one of the 2-week normal E D L 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 to the antiserum used in the RIA.  125  I - P V binding  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). Calbiochem  Calmodulin (16,723 daltons) was purchased from  whereas CaBP was purified  Physiology, from bovine cerebellum.  by Dr. Baimbridge, of  the  Department of  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  Chloramine-T technique of Greenwood and Hunter (1963).  according  to  the  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 125  I (Amersham), and 10 pi of Chloramine-T (5 mg/ml buffer).  on ice and the reaction was allowed to proceed for 2 min.  This solution was kept  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.  parvalbumin had a specific radioactivity of 3 Ci/m mole. 70  The resulting  1 2 5  I-  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 ( I) 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 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. 125  125  71  cm  RIA  fafo  Antigen & Antibody in muscle sample  Incubat ion  -"O  Add Radioactive Label (  ->  -D3  125  l)  Incubation  Separate bound and free fraction with 2°Ab  fe ^4  fe fe  Count radioactivity bound to immunoprecipitate  ^4  7Z  All  assays  were  performed  dilutions  were  made  in  contained  8  h e a t i n g , to 2000  ml  mixing  g  which  with  RIA  barbituric 41.2 g o f  distilled  triplicate buffer  acid  The  and  100 p i  (rat  skeletal  reaction of  water  (pH  muscle  was used i n  from  a dilution  in  1000  series o f  of  The  working  pi  dilution  ranged 240  7.5  was  EDTA  100  80  pi  pi.  ice.  All  barbital  buffer  water,  while  was brought prepared  to by  and 0.02% sodium  of  ng/lOOpl  ng/100  on  then  antiserum  series used f o r  from  to  stock  volume  buffer  buffer,  kept  distilled  the  BSA, 1 m M  of  The  The  ml  added and  700  Calbiochem)  glass tubes  as f o l l o w s .  with 6 g / L  sample.  cm  were  8.2-8.5).  contained  standard or  10x75  prepared  sodium barbital  mixture  either  in  dissolved  250 m l of stock barbital b u f f e r  azide.  CaBP  of  the  in  the  to  (1:400),  PV  0.156  standard ng/lOOpl.  C a l m o d u l i n was used  in  d i l u t i o n s o f 1 p g / 1 0 0 p i to 3 1 . 2 5 n g / 1 0 0 p i . Maximum either only pi  standard buffer.  of  for  standards Bound  The  vortex  24  sample. assays  Tubes  were  were  curves  until  The  the  were  for  nonspecific  cpm/tube). designed  added  and  overnight  on  of  was with 125  I  relationship plotted  on  test  same  at  day  1  P E G (20%  drained a on  by  stream a  the  and  then  tap  Beckman  between  percent  semi-logarithmic  and  in  paper.  the  pi The  at 4 0 0 0 tubes  while  binding  of  to  and  x  the  Percent  c o u n t s ( s t a n d a r d o r s a m p l e ) - N S B x 100 total counts  cold the  at  4°C.  gamma  tubes  a  were  sink.  and  counter.  amount binding  the  g a n d 4°C  over  gamma  100  bovine  assay  inverted  of  antiserum  of  the  1180  returned  100  centrifuged  model  addition  of  as:  73  by  of  contained  overnight  2  water  100 p i  incubated  dH 0).  of  and  measures  followed  addition  inversion  of  (NSB)  cross-reactivity  by  of  4°C  buffer  assay was t h e n  separated ml  800 pi of  binding  The  to  the  appeared cloudy  gently  detection  showing  parvalbumin  saline) they  RIA  were  supernatant  washed to  were  tubes c o n t a i n i n g  incubated  (10,000  the  fractions  mg/ml  mixed  prior  For  label  free  (8  min.  tubes  hr.  and  and  globulin  dry  or  was measured i n  labelled parvalbumin  (4°C)  30  binding  allowed  for The to  Standard  of  unlabelled  was  calculated  SPECIFIC  METHODS  Immunohistochemistrv Normal killed  by  (+/+)  cervical  amputation and  and  twitch half  soleus  was  described  content,  muscles  cryostat  Due  to  sections  embedding  the  procedures  plastic-sections  were  semi-thick  were  Epon,  was  not  achieved  was  with  a  of  any  to  through  the  weeks  were  correlate  digitorum  parvalbumin,  an  this  longus  the  or  and  attempt  correlate  The  pm)  staining  most  cumbersome  and  Penetration  and  precise  each  staining postfixed  fixation  and  Some of  thin  (gold  to  the  silver)  ultrastructural  the  antibody  unreliable,  localizations  lengthy  the  slow-  and  or  IV.  the of  was  by  and  regions  unfixed  were  fiber-type  (EDL)  Table  (1  the  obtained  Many  in  age  histochemical  use o f  semi-thick to  of  muscle  mid-belly  as o u t l i n e d  protein.  further.  somewhat  32  immunohistochemistry.  in  poor  and animals  attempt  investigated  of  2  immunohistochemical  for  PV  localization  pursued  an  cut  for  at  dystrophic  a n d alternate  for  mice  extensor  out,  either  )  In  fast-twitch  initially  plastic-sections  2 J  week  solubility  stained  immunohistochemical  /dy  above.  unsuitable were  2 J  2  dissected  high  was  was e m b e d d e d i n  method  the  were  (dy  The  processed separately  procedures.  tissue  dislocation.  procedure  parvalbumin  dystrophic  so  of  and  in  the  that  this  parvalbumin  freeze-substitution  fixation  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  preliminary  conducted and  on  mouse  Heizmann,  screening  and  modification Muscle  of  halves  immediately vials  were  was  slowly  test  of  the  the  antisera  cerebellum  1981). on  the  A  suitable  ELISA  unlabeled  were  immersed surrounded dehydrated  frozen into by  which  is  known  The  antibody  vial  of  dry  ice  and  paraffin  to  be  from  positive  1:10  for  was  technique  cooled  by  picric/ethanol  then  stored  embedding 74  at  fixative  according  of  parvalbumin based  Sternberger  kept  the  2  at  was (Celio  on  used  nitrogen  for to  1:3200)  method  liquid  -70°C  to  chosen  immunohistochemical  enzyme-linked  1%  range  concentration  isopentane  a  for  (dilution  antisera  test.  in  titer  this  was  a  (1979).  and  then  -70°C.  The  The  tissue  weeks. following  steps:  (a)  three  changes  chloroform Paraffin dried  at  2  either  for  2  ethanol  4°C,  sections  subsequent H 0  in  (c)  (10  for  immersion  um  rehydration  in  a graded  min,  washed  (Miles)  against  mouse  parvalbumin  at p H  goat  4°C.  in  were PBS  for  90  with min  prepared were  in  was  containing  min  in  100 p i  3%  in  to  to  X-100,  with  H 0 ,  40°C.  the  sections  at  and  deparaffinization  and  blocked  in  5%  1:400  in  10%  normal  goat  The  dilution  antiserum in  3% N G S ) and incubated overnight washed  temperature After  in  for  PBS  90  for  15  in  the  min  another  15 m i n  anti-peroxidase  room  containing  10 m i n ,  Both  goat  serum.  buffer  (TB)  at  after  2  (Sigma),  which  the  antibody  in a  moist  they  were  second  wash in  normal  Tris-HCl  min,  (PAP)  temperature.  in  temperature.  glass  staining.  a  hr  slides,  incubated  non-specific  6-12  room  sections were  then  2  about  After  the  diaminobenzidine/H 0  for  2  at  at  for  albumin-coated  ethanol,  were  5 mM  immersion  chloroform  on  hr  peroxidase  buffer in  in  reduce  room  (b)  P B S , and  c h a m b e r at  a rinse  2  series o f min  rabbit  antibody  developed  hr  24-48  sections  a moist  by  1  4°C,  collected  applied  at  at  (1:100, M i l e s ) .  the  the  followed  reaction  30  chamber  ( G A R ) IgG  incubated  Cappel)  30  the  for  for  for  was  After  a moist  anti-rabbit  were  or  7.3 ( P B S , 0 . 3 % T r i t o n  at  incubated  for  hr  were  60°C  (NGS)  chamber  thick)  at  serum  buffer  12-24  hr  15  2  over  antibody,  P B S , sections  complex  the  (1:100,  G A R and P A P Final  pH  7.6.  30  mg/100  washes  The  enzyme ml  sections were  in  PBS  rinsed  in  T B to s t o p t h e r e a c t i o n . The in  sections  xylene,  were  and  also  identically  Antiserum  used  as  then  dehydrated  coverslipped  processed  animal.  were  a  with  and  preabsorbed  control.  through  Cytoseal  60  simultaneously overnight  Sections  were  an  (Stephens  with  with  a s c e n d i n g series o f Scientific).  preimmune  purified  examined  and  ethanol,  rat  serum muscle  cleared  Control from  slides  the  same  parvalbumin  photographed  on  a  was Leitz  Orthoplan photomicroscope using T e c h n i c a l P a n 2415 K o d a k f i l m .  Histochemistry A  modified  method  for  myosin  ATPase 75  staining  was adopted  because of  its  ease  of  application  (Doriguzzi  consuming  than  ammonium  sulfide,  et  a l . , 1983).  previously and  This  published  all  three  method  has  the  protocols.  advantage  It  extrafusal  also  fiber  of  being  eliminates  types  could  less  the  be  time  use  of  distinguished  s i m u l t a n e o u s l y w i t h this p r o c e d u r e using the same p H . The  respective  processed  for  isopentane Serial to  the  EDL  immunohistochemistry,  were  cooled  60  sec (two  the  ATP  basic  rinsed  ethanol  at  (Stephens  and  embedded  and  thick)  soleus  stored  were  staining.  The  method  room  temperature  for  5-6  with  the  distilled  100%)  for  min  basic m e d i u m .  sections  were  2 min  Scientific).  stained  and  min  xylene, and  taken  for  sectioning.  and  on  air  then  dried  coverslips  washed  for  at  twice  37°C  20  sec  in  mounted a  in  myofibrillar  The  rinsed  those  frozen  for  blue  then  to  and  incubation  toluidine  were  each, cleared in  photomicroscope using T e c h n i c a l P a n 2415 K o d a k  coverslips  4.6,  a 20  1%  were  required  glass  pH  Coverslips  Photographs  liver  D o r i g u z z i (1983).  at  in  mouse  were  After  rinsed  water.  of  corresponding  until  on  sections  the  the  fresh  -70°C  collected  a c c o r d i n g to  in  muscles,  in  at  4.6  medium,  (70, 90 and  um  before  changes)  well  nitrogen,  (10  pH  preincubated  for  60  at  of  liquid  temperature  calcium-ATPase  then  by  cryostat-sections  room  were  halves  in and  ascending in  Leitz  Cytoseal Orthoplan  film.  Statistical A n a l y s i s Muscle myosin  fibers  ATPase  fiber-types the  case  commonly  the  used  1/slow-twitch  for  counts  a  categorized They  were  Camera Lucida  myosin  ATPase  classifications oxidative  2B/fast-twitch, fiber  staining.  using of  were  L  intercomparisons  parametric of  normal  designated  staining,  =  oxidative-glycolytic  included  counted  attachment  (Brook  (SO),  and  and  this  after  light to  a  (L), Leitz  Kaiser,  1970;  2A/fast-twitch,  (FOG).  Analysis  and  of  variance  dystrophic 76  immunohistochemical  dark  group  of  (D),  bright  nomenclature  type  analysis  PV  field  et  al.,  glycolytic data  (ANOVA) means.  intermediate microscope.  corresponds  Peter  the  or  to  D  and  generated  The  the  1972):  (FG),  and  and  from  In more  = I  (I)  =  type type these  Tukey's  procedure  statistical  computer  programs  in  the  Dynamics  were  "Stats used  Plus"  for  and  ANOVA  computations  packages  according  supplied  to  by  standard  Human  statistical  System  procedures  ( K l u g h , 1974).  Determination of Parvalbumin Content i)  Protein  determined Rad  in  method  Determination. muscle  using  extracts  porcine  week  normal samples were  week  dystrophic  Total  soluble  corresponding  gamma run  globulin  o n the  samples required  to  the  protein those  as  a  used  for  standard.  standard (20-140 Bio-Rad  (TSP)  pg)  concentrations  the  Both  RIA  the  microassay (1-20  All  20 m M  at p H 7.4 ( 1 0 0 m M  NaCl,  1  DTT,  azide).  tubes  vortex, The  1 mM  without  protein  Philips  PV  frothing,  and  concentration 8600  0.02%  sodium  were  was  allowed  to  determined,  spectrophotometer  and  at  the  The  sit  at  TSP  assay room  optical  32-week  pg).  i n T r i s saline b u f f e r  EDTA,  the  2-  the  2-  standards  were  temperature  density  at  concentration  Bio-  and  B i o - R a d assay, w h i l e  samples were made up mM  by  were  and  Tris  base,  shaken for  595  (O.D.  was  read  on  30  5 9 5  ),  a  min. on  from  a the  s t a n d a r d c u r v e ( F i g s . 21 a n d 2 2 ) . ii)  Radioimmunoassay.  muscles  was  measured  by  was  developed  in  was  from  sources.  two  Calbiochem initial  and  dilution  muscle  was  160 n g / initial  also  100 p i dilution  our  32  used buffer. was  parvalbumin  content  radioimmunoassay.  laboratory.  u s e d at of  The  The  Purified  rat  as a In  of  a  skeletal of  whole  standard.  to  muscle 80  This  dilution  E D L a n d soleus  anti-mouse  parvalbumin  antiserum  used  standard  the  assay  purchased  from  as  a  parvalbumin pi  homogenate  assay, the  compare  32 a n d 2 w e e k  ng P V / 1 0 0  muscle  a preliminary  found  Rabbit  parvalbumin  an initial dilution pl/ml  in  buffer. from  represented standard  favourably  to  was  a  that  In  of  addition,  mouse  PV  curve  in  fast-twitch  concentration  produced the  an  using  purified  of this rat  parvalbumin. The buffer  m u s c l e samples that  were  further  diluted  were to  50  originally times  homogenized in  volume 77  in  the  Tris  a 20 saline  times  volume  buffer  of  used for  Tris the  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 . Total p r o t e i n values f o r a l l the 3 2 - w e e k m u s c l e samples w e r e e x t r a p o l a t e d f r o m this c u r v e .  78  soluble  4  0  60  80  Protein  Concentration  7<?  100 ug/100 ul  120  140  F i g u r e 22 Standard curve for 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 total soluble protein i n n o r m a l a n d dystrophic 2 - w e e k muscle samples.  80  to  calculate  the  3  -  6  9  PROTEIN  12 CONCENTRATION  81  15 ug/100ul  18  21  protein  assay.  The  samples  were  further  diluted  in  RIA  buffer  and  tested  in  the  section  for  following concentrations:  All  assays  were  N o r m a l 32 w e e k E D L  1:100,  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 soleus  1:5, 1:10  D y s t r o p h i c 32 w e e k soleus  1:10, 1:20  A l l 2 week samples  1:5 -  run  in  triplicate  as  described  1:200  1:160  under  the  general  methods  the R I A . Parvalbumin ng/100  pi  value  content for  in  the  parvalbumin  muscle at  a  sample  given  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  82  was  percent factor.  calculated  binding  from  by  extrapolating  the  standard  a  curve,  TABLE IV. Summary of fixation and sectioning immunohistochemical localization of parvalbumin.  Embedding/ Fixation 10% Ca++-Formalin acetate - perfusion fixed -  post-fixed  methods  investigated  Morphology Status  Permeability to A n t i b o d v  Staining  cryostat, -floating  -a  +  +  and adhered to s l i d e s - paraffin  -a  +  Sectioning  f r e s h tissue - acetone or ethanol post-fixed  cryostat  —  +  5% acrolein  cryostat paraffin Epon  -a +b  ++ + +  2.5% glutaraldehyde i n 0.1 M S o r e n s e n ' s buffer  Epon  -b  0.5% Benzoquinone in P B S buffer  cryostat paraffin Epon  Bouin's  paraffin vibratome  +  cryostat Epon vibratome  + +  -c  paraffin  -  -  -  paraffin  +d  ++  ++  in P B S buffer  fixative  4% paraformaldehyde/ 0.1% glutaraldehyde i n 0.1 M S o r e n s e n ' s buffer freeze-dried/ acrolein vapour-fixed freeze-substitution 1% p i c r i c a c i d i n e t h a n o l at - 7 0 ° C  —  —  + +  + + +  — —  +  a b c  p o o r , + m e d i o c r e , ++ g o o d s h r i n k a g e o f the tissue u p o n f i n a l d e h y d r a t i o n w r i n k l e s i n t h e E p o n d u e to e t c h i n g p r o c e d u r e 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 tissue b l o c k s :  d  sections too t h i c k s o m e tissue a r t i f a c t c a u s e d b y either too s l o w f r e e z i n g or a l l o w i n g t i s s u e to d r y o u t b e f o r e e m b e d d i n g  83  +c  +  -  for  RESULTS  Characterization of The shown  electrophoretic  properties  be  those  to  1982;  similar  Heizmann,  parvalbumin that  Parvalbumin  rat  (Jasch  normal  Moase,  of  The  with  muscle the  1985).  soleus samples  rat  In that  mouse  skeletal  skeletal  muscle  comigration  EDL  comigrates  and  dystrophic  1984).  with  PV  to  of  of  samples, resolved band  addition,  rat  also  on  identified PV  believed  parvalbumin  parvalbumin  experiments  protein  was  muscle  as  comigrated  to  be  PV  rat  gels  PV  in  et  skeletal  (Fig. a  with  (Fig.  been  (Heizmann,  pure IEF  have  muscle  23),  showed  previous  the  band  24A).  al.,  report in  the  This  band  was  IEF  (Fig.  25)  not e v i d e n t i n m u s c l e samples f r o m the n o r m a l adult soleus ( F i g . 2 4 C ) . Comparison showed  a  of  definite  decrease  32-weeks  of  the  dystrophic  adult  altered detail  in in  age.  the a  experiments  A  s o l e u s at  in  stage.  particular,  genotype,  the  but  it  study  increase the  same  (Jasch normal  protein  PV  band  undetectable  whole  of  the  PV  normal  age.  A  these  and and  have  Moase,  between  was  either  both  normal  this  and  (Fig.  EDL or  at  band  in  discussed similar  26)  dystrophic  EDL  proteins  comparison,  samples  and  in  muscle  identified  muscle  the  dystrophic  noted  other  By  normal  by  the  was  of  been  in  in  levels  1985).  prominent  samples  band  number  dystrophic  in  muscle  above  but  distribution  the  was  density  muscles  2-week  difference  dystrophic  the  dramatic  previous the  and in  dystrophic  of  In  normal  showed  animals  samples  at  regardless  dystrophic  soleus  were in IEF no this of  muscle  samples. The  positions  two-dimensional Moase, the  1985).  proteins  2-week weight  the  by  labelled  in  band  of  Figures  confirmed This  mouse  other  Comparison  samples range.  gels  of  that  skeletal  muscle  investigators our 27 PV  gels -  to 30  (Crow those  of  migrates  was c o n f i r m e d  proteins  to 84  the  and  2-week  be P V b y  been  Kushmerick,  published  as a s i n g l e  have  was  the  RIA  to  1982;  Jasch  basis f o r  samples. band  characterized  14  K D  as d e s c r i b e d i n  the  and  identifying  Examination the  on  of  the  molecular methods.  F i g u r e 23 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 g e l s 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 r a t 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 t h e m o u s e m i g r a t e s to t h e s a m e i s o e l e c t r i c p o i n t as p u r e r a t 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 t u d i e s o f these samples have been reported earlier b y Jasch a n d M o a s e (1985). I E F gels are o r i e n t e d w i t h t h e b a s i c e n d to t h e l e f t a n d t h e a c i d i c e n d to t h e r i g h t . M o u s e muscle samples are f r o m n o r m a l 3 2 - w e e k - o l d animals.  85  \  1. mouse  HUH 2.  rat  —  PV  1 > ' • » 3 PV  i  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 bands o f n o r m a l ( N ) a n d d y s t r o p h i c ( D Y ) adult E D L a n d soleus ( S O L ) muscles separated a c c o r d i n g to t h e i r isoelectric point on isoelectric focusing tube-gels. The 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 a n a r r o w . N o t e the a p p e a r a n c e o f this b a n d i n the d y s t r o p h i c soleus a n d the decreased d e n s i t y o f the P V b a n d i n the dystrophic EDL. I E F g e l s a r e o r i e n t e d w i t h t h e a c i d i c e n d to t h e l e f t a n d t h e b a s i c e n d to t h e right.  89  ©  F i g u r e 26 Whole muscle protein distribution, isolated by isoelectric focusing tube gel electrophoresis, 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 soleus ( S O L ) samples. T h e p o s i t i o n o f t h e 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 t h e a r r o w for each gel. A t 2 w e e k s o f age o n l y the E D L samples c o n t a i n detectable quantities o f parvalbumin. T h e d e n s i t y o f t h e b a n d a p p e a r s to b e 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 samples.  91  91  The 27  PV  spot  and  The  28)  was but  prominent barely  distribution  of  in  detectable  proteins  adult  muscles (Jasch and  been  attained  by  2  the  at  weeks  in  2  Moase,  gels  of  normal  either  weeks  of  of This  age;  however,  of  the  age  1985).  and  dystrophic  soleus  was  would  EDL  extracts  similar  to  (Figs.  that  indicate  that  adult  protein  the  samples 29  (Figs.  and  reported  30).  for  the  profile  has  concentrations  are  the  adult  probably a c h i e v e d later d u r i n g postnatal development.  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 The  titre  described soluble (Fig. As  in  can  from  gel  molecular  methods,  then  seen  the  antisera  proteins  and  be  the  the  muscle  31a)  of  to  were  the  mouse  resolved  transferred  31b,  all  of  nitrocellulose.  markers;  PV  was  monospecificity  initially  were  Figure  the  weight  and  they  in  against  however,  to  the The  the  tested  by  by  by  immunoblotting  one-dimensional  nitrocellulose low  ELISA  paper  corresponding  lane lane  in in  and  SDS for  molecular-weight  left-hand  procedures, RIA.  The  electrophoresis immunoblotting.  proteins  Figure  the  as  transferred  31a  half  of  contained  the  gel  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 pg o f rat m u s c l e P V . As  illustrated  proteins  present  most  intensely  used  as a  intense in  the  in to  the  the  muscle rat  and  the  soleus  indicated  the  32  and  PV  normal EDL  by  34a,  extract.  muscle  dystrophic  normal  reaction  Figures  the  control,  for  in  In  control,  EDL  and  of  antisera  the  adult  the  not  As  PV  though  the  protein  reacted  was  also  staining  was  less  was  31a), in  other  which  PV  (Figs.  any  antisera  e x p e c t e d , the  Even  of  with  gastrocnemius  electrophoresis  amounts  react  s a m p l e s , the  soleus samples.  trace  did  normal  ( F i g . 32).  one-dimensional  presence  PV  undetected  the  the  antisera  slow-twitch  muscles regardless o f genotype ( F i g . 32). The serum standard  results  with  of  CaBP  curves  in  the and  Figure  RIA  designed  calmodulin, 33.  Only  to  and the  test thus  PV  93  the its  standard  cross-reactivity monospecificity, resulted  in  of are  complete  the  anti  depicted  PV as  displacement  F i g u r e s 27 a n d 28 Two-dimensional electrophoresis patterns of total muscle proteins separated according to t h e i r i s o e l e c t r i c p o 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) and dystrophic ( D Y ) E D L muscle samples. E a c h I E F gel contained proteins derived f r o m 5 m g wet weight muscle. I E F w a s r u n w i t h t h e b a s i c e n d to t h e l e f t a n d the a c i d i c e n d to t h e r i g h t . T h e prominent p a r v a l b u m i n band ( P V ) appears i n both normal a n d d y s t r o p h i c s a m p l e s at a p p r o x i m a t e l y 14 K D . A c t i n (A), tropomyosin ( T M ) and m y o s i n light c h a i n s ( L C ) are also i n d i c a t e d .  94  2 WEEK N  EDL  ©  IEF. SDS  A/  L  MJTM  C  l  f ^  ,  ;  LC2  f  <~  2  WEEK  V  DY EDL  L  C  3  f  (2^  IEF  *  SDS  TM LC1  f  _^LC2  95-  f  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 patterns o f total muscle proteins f r o m normal (N) and dystrophic ( D Y ) 2 - w e e k s o l e u s , s e p a r a t e d 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 w a s f r o m l e f t ( b a s i c ) to r i g h t ( a c i d i c ) . The 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 patterns.  96  2  WEEK  N SOLEUS IEF  SDS  TM LClg^  TLC2  s  ^LC2  f  PV  ©  2 WEEK DY S O L E U S IEF SDS  A  TM LC1  S  TLC2 PV  V  s  l£2f  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 adult m u s c l e samples f r o m w h i c h the proteins 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 slab gel i n F i g . 3 1 a represents the c o r r e s p o n d i n g h a l f o f the gel that was stained with Coomaisse Blue R250. T h e a r r o w indicates 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 s o l e u s ; e) n o r m a l s o l e u s .  F i g u r e 32 Immunoblot ( F i g . 32) o f normal and dystrophic adult muscle homogenate samples, t r a n s f e r r e d f r o m the S D S slab gels ( F i g . 31b) The immunoblot is r e v e r s e d to t h e pattern s h o w n i n F i g . 31a, w i t h one exception. T h e extreme l e f t - h a n d lane i n F i g u r e 31a contains m o l e c u l a r weight markers while the c o r r e s p o n d i n g lane 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 ) are 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 a n t i s e r a .  98  99  of  1 2 5  I-PV  binding  to  the  antiserum.  No  cross-reactivity  with  CaBP  or  calmodulin  was o b s e r v e d . The altered  distribution  in  the  of  some  dystrophic  for  the  adult  evident  that  the  distribution  to  of  soleus  the  resembles  bands.  Only  characterized  muscle  normal that  PV in  of  higher  at  32  of  present  on  study,  shows  examination  of  the  dystrophic  obvious,  the  pattern  gels.  but  34  in  muscles  the  soluble-proteins  Figure  close  less  fast-twitch  weight  age.  (arrowheads)  Although  identified  of  Upon  proteins  the  molecular  weeks  samples.  soleus.  was  the  the  muscle  pattern  that  of  with  The  probably  respect  other  the  gel,  EDL  it  is  the  is  similar  dystrophic  bands  various  also  banding  particular  prominent  represent  the  this  in  to  was  protein were  myosin  not  light-  chains, tropomyosin, troponin and actin. Whereas seen  in  week  the  definite adult  extracts  samples.  revealed  samples  (Fig.  35a).  due  (Fig.  PV  the  identical  differences  resulted  EDL  in  preparations,  Although  differences  changes  to  and  some  S D S gels features  could  be  genotype.  of  the  corresponding  when  comparing  The  and  reacted  Parvalbumin  was  undetectable  muscle  and  seen b e t w e e n  35b),  other  PV  band  positively in  fast  to  the  either  of  immunoblots normal  and  was  could of  and  after  soleus  the  2-  muscles,  identifiable  antisera  be  dystrophic  slow-twitch  clearly  the  proteins  in  no  both  immunoblotting  samples  by  SDS-  P A P methods  using  P A G E ( F i g . 35b), but was f a i n t l y evident after i m m u n o b l o t t i n g ( F i g . 35a).  Immunohistochemistrv The the  antiserum  fixed groups were  distribution  by  against  of  PV  mouse  in  skeletal  freeze-substitution.  of  staining  considered  intensity: PV to  PV.  category  in  Table  is  given  fibers  muscle  Parvalbumin light  positive,  immunoreactivity  muscle  (L),  was  PV,  investigated  on  paraffin  immunoreactivity  dark  (D),  whereas  the  A  summary  of  V.  Muscles  were  100  and  light the also  by  sections was  mean  were  The  (I).  assumed  percentage  fiber-typed  muscle  categorized  intermediate fibers  of  for  of  tissue  into  three  dark  fibers  to  fibers  myofibrillar  have in  no each  ATPase  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 Gradient (5-22%) one-dimensional S D S - P A G E of 32-week normal (N) and dystrophic ( D Y ) m u s c l e proteins f o r gastrocnemius ( G A S T ) , E D L a n d soleus. T h e p a r v a l b u m i n b a n d (PV), approximately 14 K D , is i n d i c a t e d b y t h e a r r o w . T h i s g e l c o r r e s p o n d s to t h e immunoblot in Figure 31. 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 also 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 bands (arrowheads). N o n e of w h i c h bands reacted w i t h the p a r v a l b u m i n antisera o n the i m m u n o b l o t . Molecular weight markers are 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 left c o l u m n .  103  /Of  F i g u r e 35 Immunological analysis of 2-week normal (N) and dystrophic (DY) whole muscle homogenates after separation on S D S - P A G E ( F i g . 35b) and electrophoretic transfer to nitrocellulose paper ( 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 b e b l o t t e d , as a c o n t r o l f o r t h e a n t i s e r a . 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 nitrocellulose membrane (Fig. 35a) is reversed to that seen in the slab gel. Nitrocellulose was incubated w i t h a n t i - m o u s e p a r v a l b u m i n antiserum. The parvalbumin b a n d is i n d i c a t e d b y t h e a r r o w . Both normal and dystrophic samples show similar 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 a g e . M o l e c u l a r w e i g h t m a r k e r s are i n d i c a t e d in 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 . 35b).  105  lOlo  and divided into L , D , and I staining groups. The  original  simultaneously  intent  type  the  Unfortunately  due  sections,  typing  not  fiber  possible.  muscle  for  to  in  using  same  from  However,  each staining  for  with  serial  the  corresponding  fibers  problems  T h e s e results are g i v e n i n T a b l e V I .  PV  tissue  sections  fiber  procedure  and  Sections f r o m  the  of  each  muscle  was  and  myosin  ATPase.  immunoreactivity  shrinkage,  of  typing  halves  freezing  corresponding  was  halves  performed  e x p r e s s e d as t h e  artifacts,  on  of  each  sections  mean  and  muscle  from  percentage  loss  the  of  to  of was  same  fibers  in  each group. i)  Adult  mosaic could  pattern be  of  PV  identified  diameters. of  EDL.  The  extrafusal  immunoreactivity  that  largest  fiber  varied  types  that  were  (35.78%)  were  intermediate  of  however,  this with  (Heizmann  et  as  type  that  al.,  2-fast  in  both  was  not  reported  1982).  fibers,  the  the  staining  further the and  light  fibers  Three  PV  fibers was  also  and  of  the  type  in  fiber  types  cross-sectional  highest of  these  percentage  small  fibers  extrafusal  fibers  intermediate  pattern  of  1982)  and  variety,  was  ICRZ  the  in  mouse  PV-positive  and  A  fibers;  staining  c a t e g o r i z e d the  1-slow  heterogeneous  cross-sectional diameter.  Heizmann,  H e i z m a n n (1982)  the  remaining  This  and  and  population  The  observed  (Celio  a  a  extrafusal  constituted  intensity  be  major  immunoreactivity  subdivided.  rat  Celio  muscles contained  antisera.  could  in  to  There  to  EDL  36).  darkest  (48.92%).  intensities  group  agreement  and  non-reactive  staining  (Fig.  according  diameter  (15.17%)  gradient  normal  fibers  intermediate  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 . When 37)  both  had  decreased.  as  was  the  the  immunoreactivity  number The  of  therefore,  was  a  greater  showed  of  profile  darkly-stained  proportion  percentage  EDL  Parvalbumin  this  of  fibers  variability  immunoreactivity  in  compared  fibers  (31.42%)  non-reactive  in  an overall  was  the  was not  light  intermediate  decrease i n  fiber  or  size  in  PV  107  and  the the  fibers  group  dystrophic intensity  (24.17%)  (44.55%).  immunoreactivity.  the  s p e c i f i c to  to  sections  fiber  from  s i z e as i n  the  EDL of  was  staining increased  The In  dystrophic  addition,  dystrophic normal  (Fig.  there  muscle.  EDL.  As  F i g u r e s 36 a n d 3 7 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 (Fig.37) EDL muscles from 32-week-old mice. Muscles were fixed by freezes 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 . Three p r o m i n e n t staining intensities can be i d e n t i f i e d : large d i a m e t e r , d a r k f i b e r s ( D ) , fibers o f i n t e r m e d i a t e s i z e 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 ) . There are a greater 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 alteration 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 its 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 fascicles d i d not stain. x400  108  (09  can  be seen  for  PV.  in  Figure  The  significant  37, small-diameter  Tukey's  interaction  statistical  effect  fibers  analysis  between  m a y be either  procedure  genotype  for  and  PV  L,  D,  or  I  in  intercomparisons staining  pattern  staining  indicated  for  a  the E D L  sections. These (Figs.  38  fiber  a n d 39) i n  intermediate the  fibers  smallest  staining (Table light  groupings  and  pattern VII).  after  which were  darkest  myosin  large  the  largest  type  2  fibers  was almost The  corresponded  a  fast  ATPase.  to  the  myofibrillar  a n d palest  fibers  with  fibers  both  fast  ATPase  were  type  and  slow  were  of  the  slow  variety.  The  reverse  to  that  seen  with  PV  type  fibers  T h e small  were  type  dark  the  after  1 slow  PV  fibers  staining  2-fast  profile  fibers,  the  characteristics,  and  myofibrillar  ATPase  immunohistochemistry  antisera  were  treatment  non-reactive  and  for  PV  but were d a r k l y stained b y the m y o s i n A T P a s e procedure. In and  this  myosin  gradation  study  ATPase  of  PV  reactive  to  evident  when  ATPase  staining  could  intensities  percentage  into  the  second  of  the  small  the  categorized  into  When  fibers the  reactivity  fell  D/type as  the n o r m a l  a  type  typing into  the the  2 group basis  VI,  fibers  than  type  slow  fibers  according 2  and  PV  L/type  was observed using  E D L appear  In  1  there  ranging  the  that  types to  immunoreactivity  contained  both  fiber  PV  P V whereas  fibers  from  comparison.  and dystrophic  2  respectively)  distinct  I/type  for  of 1  between  contained  obtained  and  three  fibers  class  percentages V  correlation  2 fast  in  (Tables  immunoreactivity. of  A l l type  procedures  be  distribution,  None  comparing  percentage  was not a complete  staining.  intense.  not  ATPase  there  from  PV.  extrafusal  based  on  groups  words,  to be s l o w e r  types  a  and  based of  fibers  their  localization,  was  myosin  PV  greater a  the c o r r e s p o n d i n g  other  nonIt  P A P and  the  was a  lower myosin  on  PV  muscles  than  is i n d i c a t e d b y t h e m y o s i n A T P a s e l o c a l i z a t i o n . The  adult  fibers  and a  typed  according  dystrophic  concomitant to  myosin  E D L ( F i g . 39) s h o w e d an increase i n the n u m b e r decrease ATPase.  in  the  number  Although 110  not  of  L/type  visible  2  extrafusal  in Figure  of  I/type  fibers  3 9 , there  2  when  was also  F i g u r e s 38 a n d 3 9 Histochemical demonstration of myofibrillar A T P a s e at p H 4 . 6 i n f r o z e n s e c t i o n s o f normal ( F i g . 38) and dystrophic ( F i g . 39) adult EDL muscles. Three staining intensities are noted i n the e x t r a f u s a l fibers: 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 outer, capsule a n d B a g 2 intrafusal f i b e r (asterisk) o f a muscle spindle cut through 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 stained i n the d y s t r o p h i c E D L . A l s o note 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 diameters i n the d y s t r o p h i c m u s c l e . x650  111  an  increase  in  intermediate of  muscle  the  number  fibers  was  of  D/type  consistent  spindles, presumably  with  the  1  fibers.  the  PV  bag2  An  increase  results.  fiber,  was  One  darkly  in  of  the  the  stained  proportion  intrafusal  for  myosin  of  fibers ATPase  r e a c t i v i t y as w a s t h e t h i c k o u t e r c a p s u l e ( F i g . 3 9 ) . ii)  Adult  antisera and  (Fig.  a  fibers.  In  (35.11%)  and  soleus.  sections  positive.  predominantly  population  of  normal  M u s c l e sections o f  were  percentage  dark  the  40)  small  extrafusal the  Soleus.  of  the  up  of  intermediate  (44.34%)  fibers  to  greater  the  dystrophic  there  was  a  large  soleus ( F i g . 41),  (26.64%),  appeared  adult  (14.60%)  fibers  There  Overall,  made  normal  intermediately  dystrophic  light  from  both  the  be  and  a  muscle, greater  an  fibers  and  (8.39%)  increase  these  proportion  darkly  in  of  <.01)  fibers  fibers  stained  of  compared  were  positive  PV  decrease  percentage  when  the  (76.73%)  smaller-diameter  small  of  (p  the  observed  number  with  non-reactive  a significant  were  and  soleus treated  both to  the  fibers  usually  for  PV  in  in  PV-  in  the  in  the  d y s t r o p h i c soleus t h a n i n the n o r m a l soleus. The extrafusal  most fibers  percentage  of  identical. fibers  striking of  the  fibers  Unlike  either  observation dystrophic  categorized  their  positively  was  normal or  the  similarity  soleus  into  the  PV  and  the  dystrophic  of  the  staining  each  counterparts,  negatively  in  the  stained  for  EDL. groups  distribution PV  was  localization  of  The was  virtually  extrafusal  evenly  mean  dispersed  muscle in  the  in  the  sections f r o m the d y s t r o p h i c E D L a n d soleus ( T a b l e V ) . This 32-week evident soleus  from has to  extrafusal the  to  dystrophic  addition  of  trend  a  both  a  soleus Figures  greater the  fiber  normal  more  was 42  diameters A  in in  also and  proportion  conversion  tissue.  homogeneous  of  observed 43,  as  in  well  correlation  of  between  113  the  extrafusal  myosin  from  fibers  intensities  section  of  the  as  intermediate  staining the  population  Table  than  there  was  dystrophic  fiber  size  its a  ATPase VI,  that  greater  fiber  types  staining.  normal  soleus  and  fiber  the  It  dystrophic  counterpart. variability  than type  in  is  the  could  in  In the  section not  be  F i g u r e s 4 0 a n d 41 P a r a f f i n sections o f freeze-substituted muscles 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 (Fig. 41) adult soleus muscles. Sections were processed for parvalbumin immunohistochemistry as d e s c r i b e d i n t h e Methods. Three staining intensities 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 a p p e a r s to b e 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 greater v a r i a t i o n i n f i b e r size i n the d y s t r o p h i c s o l e u s w h e n c o m p a r e d t o its 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 sections 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) adult soleus m u s c l e s , stained for myofibrillar A T P a s e p H 4.6. T h e three s t a i n i n g intensities 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 soleus contains m a n y s m a l l diameter extrafusal fibers w h i c h vary in staining intensity. O v e r a l l t h e r e a p p e a r s 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 soleus. 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 s e c t i o n s . T h e B a g 2 i n t r a f u s a l fiber (asterisks) stains d a r k l y in both normal and dystrophic muscles, however, only the o u t e r c a p s u l e o f t h e s p i n d l e i n t h e 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  made the  since  all  three  intensities  were  observed  regardless  of  the  diameter  of  myofiber. Generally,  muscles in  staining  the  was  using  muscle  capsule observed  E D L , one  stained  of  in  .  It  myosin  less  was  either  activity  or  was  myosin in  the  (Figs.  observed  in  the  sections  ATPase  42  and  however,  fibers  typically  differences  of  in  a  PV  the  adult As  soleus muscles  The  43);  thick  this  an  outer  was  only  no  PV  had  however,  spindles.  length  the  43).  animals,  some  from  histochemistry.  sections o f  (Fig.  dystrophic  regional  the  intrafusal  throughout  possible that  in  stained  The  normal  taken  with  fibers  darkly  tissue.  sections is  intrafusal  also  fiber  evident  than  ATPase  intrafusal  serial  performed  the  dystrophic  positively-stained of  of  spindles  the  immunoreactivity  staining  for  muscle in  were  P A P immunohistochemistry  32-week  darkly  spindles  occasional  Immunohistochemical  muscle  spindle  were  immunoreactivity  may  not exist  i n t h e i n t r a f u s a l f i b e r s as w a s s e e n i n C h a p t e r 1 w i t h t h e m y o s i n A T P a s e s t a i n i n g . iii)  Two  week E D L .  EDL  ( F i g . 44)  EDL  showed  outlined  in  dystrophic Figure  45,  sections. in  the  and a  dystrophic  slightly  Table  were  the  overall  normal  PV  EDL  greater  V.  EDL  The  The distribution  The  percentage  for  adult  EDL.  of  level  in  the  At  2  although  of  light  and  of  weeks  of  the 2 - w e e k - o l d  sections f r o m (27%  fibers  in  was  higher  the  dystrophic to  both  normal  the  As  in  the  19%)  c a n be  muscles resembled  age,  muscles  did  not  as and  seen  dystrophic  fast-twitch these  normal  compared  4 1 % , respectively).  staining  neonatal  the  fibers  extrafusal  P V (50%  background  distribution  P V was similar in  (Fig. 45),  majority  positive  of  in  EDL  that  seen  show  the  v a r i a t i o n i n f i b e r diameters o b s e r v e d i n the adult m u s c l e s . Intrafusal regions  of  sectioned  fibers  muscle through  immunoreactivity  in  intrafusal  that  fibers  fibers  contained  PV  that  stained  spindles the the  of  positively  the  2-week  equatorial intrafusal  were  ( F i g . 49)  and  fibers  positive  for  for  as w e l l  PV  sections  were (Figs.  juxtaequatorial  ( F i g s . 44  and  it  appeared  as a l l  of  the  45  nuclear  one  chain  49).  by of  the the  fibers.  in  polar  Spindles  revealed  Judging that  observed  and  regions  48).  PV,  118  often  no  size  of  nuclear This  PV the bag  finding  is c o n t r a r y to t h a t r e p o r t e d i n t h e a d u l t E D L o f t h e r a t ( C e l i o a n d H e i z m a n n , The activity  distribution  (Table  Approximately fast  (type  normal  2)  or  tissue  sections,  86%  of  than  respect  proportion  of  similar the Few  and  the  from  the  typed  (Fig.  were  Sections dystrophic staining  of  of  younger  a lower  fibers  ATPase  groups  the  normal  EDL.  distribution  two  fibers  46)  was  animals,  percentage  of  and  from  were  the  muscles. pattern  the  was  intermediate  a  fibers  in  EDL  of  the  either  the  often to  stained  the  samples  muscle  that seen  adult  differed  fibers.  in  the  percentage  than  EDL.  therefore  2-week  greater  ATPase  ( F i g . 47)  compared  dark  reversed f r o m  there  and  normal  and  myosin  observed  When  of  to  dystrophic  stained  fibers  intermediate  fibers  according  lightly  intermediately-stained  2-week  the  these  in  muscle  extrafusal  myosin  from  fibers  extrafusal  those  the to  sections  slow  was  dystrophic  overall  the  VI)  variety.  darker  with  of  1982).  calculated  The  adult.  of for  In  dark  or  the  adult  normal  (Fig.  EDL. Intrafusal 46) 1  or  fibers  dystrophic  for  myosin  intrafusal  in  polar  ( F i g . 47) ATPase  fibers,  regions  2-week  under  presumably  of  EDL  acid chain  muscle  spindles  in  stained according  preincubation fibers,  were  to  either that  conditions.  the  described in  Chapter  The  diameter  unstained  while  extrafusal  muscle  small  the  large-diameter  intrafusal fibers were d a r k l y or intermediately stained. iv) normal a  Two  ( F i g . 48)  small  results were  Week  the  noted  in  weeks  of  age.  noted  in  the  in  the  and  population for  of  muscles  staining  The  distribution adult  adult little  fibers, are  of  soleus,  2  of  however,  between PV  but  in it  the was  Parvalbumin weeks  the  soleus were  compiled  pattern  soleus.  after  majority  ( F i g . 49)  extrafusal  the  normal  varies  The  dystrophic  soleus  dystrophic  therefore,  Soleus.  of  age  119  non-reactive  that  in  stained  Table  the  V.  extrafusal  and  fibers  dramatically  the  for  significant  was  very  different  soleus.  in In  in  PV.  dystrophic  immunoreactivity in  for  positively No  normal  fibers  the  the  There  was  PV.  The  differences soleus  similar  from the  both  that  normal  dystrophic  at  to  2  that  observed muscle, soleus,  F i g u r e s 44 a n d 45 Immunohistochemical localization of parvalbumin in freeze-substituted paraffinsections 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) neonatal E D L muscles. T h e three m a j o r t y p e s o f f i b e r s a r e l a b e l l 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) i n s t a i n i n g . M u s c l e spindles (arrows) are i n d i c a t e d i n b o t h sections. N o t e that intrafusal fibers 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 parvalbumin. Three more spindles (not l a b e l l e d ) a r e p r e s e n t at t h e l o w e r e d g e o f F i g . 3 0 . T h e r e a p p e a r s to b e a r e l a t i v e l y large p r o p o r t i o n o f l i g h t l y stained 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 sections o f n o r m a l ( F i g . 46) a n d dystrophic ( F i g . 47) neonatal E D L muscles, stained 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 t h e e x t r a f u s a l f i b e r s at t h i s age a p p e a r to b e 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 ( a s t e r i s k s ) 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 (arrows) 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  PV  localization  varied  with  age,  indicating  muscle  fibers  from  an  interaction  between  staining  pattern,  age a n d g e n o t y p e . Intrafusal stain  for  fibers  PV  ( F i g . 48).  which  suggested  were  that  neonatal  Polar  positive  both  muscle.  regions  for  nuclear  the  PV  of  and  more  zone  of  muscle spindles d i d  muscle spindles, however,  ( F i g . 49).  bag  Interestingly,  juxtaequatorial  The  nuclear  PV  was  diameters  chain  of  fibers  contained  these  may  occasionally observed  intrafusal  intrafusal  contain in  fibers  PV  one  not  of  in  the  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). Extrafusal soleus  could  be  reactivity. L/type 2  both  categorized  variety than  variety.  from  the  three  The  of  but  soleus.  and  dystrophic  groups  fiber  soleus c o n t a i n e d similar  fibers  mean  was  Intrafusal  to  51)  2-week  myosin  ATPase  classification significantly  percentage  lower  fibers  (Fig.  according  predominant  a  1  50)  staining  dystrophic  D/type  adult  (Fig.  the  counterpart  percentage normal  normal  genotype,  VI).  normal  the  the  into  of  (Table  its  The  sections  in  Regardless  2  fibers  fibers  than  stained  of  that in  was  more the  the  I/type  D/type  observed  a similar  in  1 the  fashion  to  t h a t o b s e r v e d i n t h e E D L at 2 w e e k s o f a g e .  Summary of Immunohistochemical Results The main  significant  effect  of  according  to  according  to  fibers  the  of  in  PV  the  the  differences stain  analysis  genotype EDL  appeared  but  than  to  vary  to  of  (more in  in  PV  an  and  interaction  variance). type the  I  myosin  in  soleus), and  according  to  of  The  fibers  ATPase  PV  genotype,  age  distribution  dystrophic age.  genotype  staining  For as  were  and  not  due  muscle  pattern  (p  appears  to  muscles), muscle (more a  well  given as  age  m u s c l e , the of  the  to  a  <.001 vary  type  D  localization  animal.  These  i n t e r a c t i o n e f f e c t s w e r e also o b s e r v e d i n the m y o s i n A T P a s e results. Parvalbumin that  made  observed  it  immunohistochemistry  difficult  using  the  to  type  myosin  the  ATPase  resulted  extrafusal method. 124  in  muscle No  a  gradation fibers true  into  of the  correlation  staining 3  intensities  distinct  could  be  groups drawn  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 sections 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) neonatal soleus m u s c l e s , stained f o r p a r v a l b u m i n . L i g h t (L), dark (D), or intermediate (I) fibers are common to both normal and dystrophic sections; however, 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 . 4 9 . 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 . 4 8 ( 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 t h e d y s t r o p h i c s p i n d l e b u t t h o s e i n t h e normal 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 of F i g u r e 49 also shows some 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 y p e s 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  for  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 3 2 a n d 2 w e e k s o f a g e . 32 Week  EDL N Light Dark Intermediate  a b  15.17 48.92a 35.78  2 Week  SOLEUS  DY 24.17 31.42 44.55  N 76.73b 8.39b 14.60b  DY 26.64 35.11 44.34  EDL N 19.00 50.00a 30.75  SOLEUS  D Y 27.07 41.00 31.98  Indicates statistical significance between n o r m a l and dystrophic v a l u e s at p < 0 . 0 5 . Indicates statistical significance between n o r m a l a n d dystrophic v a l u e s at p < 0 . 0 1 .  129  N 68.72 10.50 21.13  DY 73.34 9.46 15.99  T A B L E VI.  D i s t r i b u t i o n o f f i b e r t y p e 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  for  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 3 2 a n d 2 w e e k s o f a g e .  32 Week  EDL N  2 Week  SOLEUS  DY  N  DY  EDL N  SOLEUS  DY  N  DY  L/type2  69.88b  17.60  50.25b  18.60  85.38  86.03  68.28  54.52  D/type 1  1.50  5.10  42.37b  15.09  11.36  13.28  27.95  32.15  5/type2  28.62b  77.27  7.33b  66.33  3.21  0.63  4.08  13.30  a  Indicates statistical significance between n o r m a l and dystrophic  b  v a l u e s at p < 0 . 0 5 . Indicates statistical significance between n o r m a l and dystrophic v a l u e s at p < 0 . 0 1 .  130  T A B L E VII. Correlation of extrafusal fiber classifications for parvalbumin and myosin ATPase staining procedures. Light  Dark  Intermediate  Parvalbumin  non-reactive slow  high fast/type2  medium fast or slow  Myosin  fast  slow  fast-oxidative  ATPase  type2  typel  type2  i  131  between fibers and  PV  and  typed  fibers  the  an  myosin  type  variety  the  soleus.  EDL  the  normal  the  adult  staining  dystrophic pattern  no  distribution from  staining.  of  slow is  fibers  of  significant  that  parvalbumin  difference  when  compared  very  similar  between  were  measured  on  to  positive  their  the  than  the  was  fibers  and  soleus  observed  that  fibers  of  the As  than  with of  counterparts. EDL  using  1982).  localization  dystrophic  2  muscle  parvalbumin  the  type  non-reactive  significantly  normal  adult  of  Heizmann,  for  in  and  EDL  fact  vary  observed  1  extrafusal  and  not  type  the  types  (Celio  did  was  the  both  based  were  of  distribution  When  fiber  immunoreactivity  more  the  immunoreactivity,  observation  PV  muscles  was  PV  PV  distribution  muscles, a  the  This  contained  Whereas the  by  The  differed  proportion  stain. had  ATPase  to  higher  ATPase  1/SO  expected,  according  overall  reactivity.  myosin  determined  categorized  contained  ATPase  a c c o r d i n g to  reactive  were  myosin  the  age  in  PV  in  The  and  PV  dystrophic  soleus.  Parvalbumin Content Levels antiserum animals  of  raised in  PV,  are  determine  fact  that  used. standard  the  of  obtain  standard Figures  mouse  PV  pure  curves 52  and  was  PV  constructed  using  this  was  sample  53,  muscle  extract  assay  done  were  by  added  with  number  a  adult  system  rat rat  PV  mouse  mid-point  the  from  1 2 5  I-  muscle.  in  view  order of  assay  antiserum  muscle  the  incomplete  Therefore, in  of  2-week  skeletal  system  PV  for  and  heterogeneous  anti-mouse  the  a  sample  samples, and  calibrated  extrapolating  from  rat  specificity.  iodination,  132  the  muscle  the  from  to  skeletal  each  the  derived  for  the  for  In  muscle  of  in  PV  mouse  incubated  dilutions  all  species  available  was  for  pooled  respectively. were  the  anti-mouse were  the  relative in  using  muscles  homogenates  indicating  rat  of  standard  muscle  PV  RIA  enough  concentration rat  by  Individual  parvalbumin  iodinated  curve  Calibration  of  to  occurred,  only  The  in  and  dilutions  rabbit.  group  shown  cross-reactivity to  the  Typical  antiserum  When  in  each  immunoassay. samples  PV  the was  and  a  homogenate. the  mouse  homogenate used  to  may  were  sample  curve  determine  procedure curves  dilution  the  to  real  introduce  made  would  of  fall  the  of  the  rat  concentration  of  PV  at  some  muscle  in  mid-point  the  i n a c c u r a c i e s the extracts  linear  so t h a t  portion  standard a  relative the  of  given  of  PV  standard  This  value  dilution.  differences  amount  the  curve.  are  While  small.  present  curve.  was  Dilution  in  The  this  e a c h assay  concentration  n e c e s s a r y 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 . Tables and  VIII  2-week  and  samples,  F i g u r e s 54 a n d 5 5 . Normal The  dystrophic detected value  with the  in  in  4.5  in  significant  at  immunoblot in  was  is  the  normal  adult  less the  the  in  in  are  the  also  various  muscles of  depicted  adult  in  histograms  and  they  and  Figure  are  dystrophic  PV  (27.89  pg  PV/mg  than  that  in  54.  reasonable skeletal  in  of  pgPV/mg  the of  conversion  All are  (1.03  to  these  adult  similar  the  agreement  with  previous  et  samples.  Of EDL  differences  with  al.,  1982;  of  were  electrophoresis reports  be this  concentrations  reported  (Heizmann  the  and  dystrophic  consistent  muscle  in  could  TSP),  dystrophic the  TSP).  found  little i m m u n o r e a c t i v i t y  content  dramatic  results in  extracts  PV  greater  very  detected  the  of  times  contrast,  amount of  levels  5.3  muscle  This  These  high  was  In  resemblance  <.001.  normal  EDL  soleus  than  depicted  data,  content  values  contained  soleus muscles.  p  PV  These  pgPV/mg TSP).  times  the  protein  muscles  normal  interest  that  content  PV  the  the  respectively.  EDL  E D L (5.27  was  greater  of  summarize  A l l t h e v a l u e s a r e e x p r e s s e d as t h e m e a n P V c o n t e n t .  adult  amount  IX  and  of  Pette  PV  et  al.,  1985).  Developmental Changes Parvalbumin samples  (Table  the  normal  the  dystrophic  were  IX).  EDL  slightly  levels  at  EDL less  were  the  However, 2  weeks  (18.99  than  pg  those  highest  unlike  (19.74  pg  PV/mg observed  the  in adult  the  EDL  samples  preparations,  the  PV/mg  T S P ) was  virtually  T S P ) as  illustrated  in  for  normal  the  133  adult  of mean  2-week-old  PV  identical  Figure EDL  the  55.  (70.8%  content  of  to  of  that  These of  the  values adult  F i g u r e 52 Standard curves for the r a d i o i m m u n o a s s a y o f rat 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 (open s t a r s ) a n d m o u s e m u s c l e h o m o g e n a t e ( s o l i d s t a r s ) . T h e m u s c l e h o m o g e n a t e w a s u s e d at an initial dilution of 1:32 to s i m u l a t e the p u r i f i e d rat parvalbumin. Values for 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 samples w e r e c a l c u l a t e d f r o m this c u r v e .  134  i  1  1  1  0.16  0.31  0.61  1.23  Parvalbumin  1  1  2.3  3.0  (ng/l00ul)  135"  1  !  1  10  20  40  T 80  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 rat 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 (broken line) and mouse muscle homogenate (solid line). T h e m u s c l e h o m o g e n a t e w a s u s e d at an initial dilution of 1:32 to s i m u l a t e t h e p u r i f i e d rat p a r v a l b u m i n . Values for p a r v a l b u m i n content i n the 2 - w e e k samples w e r e c a l c u l a t e d f r o m this 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 E D L and soleus (SOL) muscles. Both the dystrophic E D L 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  lOS  AQ  10S N  103 AQ  103 N  OO o o o o o oo oo o  g  CH  m > Z  •o < SL  8 m Z  02  value)  suggesting  that  synthesis  of  PV  comparable  to  adult  levels  has  not  been  attained b y 2 weeks o f age. The amounts  RIA of  quantities content does  in  obtained  of  values  of  either  IX).  levels  those  with  as  a  were  for  the  mean  PV  different  normal  the  on  PV  normal  samples was  the  Based  the  by  2-week  expressed  coincide  The  change  the  in  were  not  age. to  PV  (Table  Minimal  for  not  or  dystrophic  minimum  these  detected  dystrophic  soleus  (Fig.  content  with  techniques  to  detectable  results,  significant in  sensitive  it  would  increase the  in  normal  s o l e u s at 55).  2  age.  the  the  appear  that  of  detect  significant  preparations.  and  not  in  both  age  32  were adult  are  the  and  in  2  muscles. weeks  virtually soleus  of  identical showed  with  early  PV  maturation  soleus  consistent  concentration  These mean  postnatal  content  dystrophic results  PV  to  value  s o l e u s at  These  study  soleus  PV  weeks  Only  enough  a  those  development  a n d m a t u r a t i o n o f s k e l e t a l m u s c l e ( L e b e r e r a n d P e t t e , 1 9 8 6 a ; K l u g et a l . , 1 9 8 3 b ) . Intergroup showed  a  dystrophic week  and  muscles  significant EDL  and  2-week  have  dystrophic  comparisons  difference the  2-week  EDL  almost  soleus  at  of  assay  weeks  EDL  muscles,  (p  <.02)  between  dystrophic results  reached 2  the  their of  age  EDL.  was adult did  not  conducted RIA  The  results  difference  significant.  concentrations not  differ  by  Student's of  between  Therefore, of  PV.  significantly  the  PV  32-week  normal  2-week  The  in  t-test,  32EDL  normal  content  or when  c o m p a r e d to t h e n o r m a l a d u l t s o l e u s . In genotype,  summary age  and  the  mean  muscle  PV  type.  content Although,  varied at  2  in weeks  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  any of  given  group  age, genotype  according and  age  to had  T A B L E VIII.  M e a n values 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  dystrophic ( D Y ) muscle samples. PARVALBUMIN CONTENT ua/ma WET WT ua/maTSP N  EDL  DY  EDL  n=5  N SOL n=6  D Y SOL n=5  M E A N  27.89 4.78  S.D.  n=6  S.E.M.  1.95  M E A N S.D. S.E.M.  0.62 0.28  5.27  1.06 0.18 0.07 0.15 0.024 0.011  M E A N S.D. S.E.M.  1.04 * 0.22 0.09  0.032 0.009 0.004  M E A N S.D. S.E.M.  4.64 0.24 0.11  0.124 0.032 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 = total soluble protein W E T W T = wet weight S.D. = standard deviation 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 IX. (DY)  M e a n values f o r p a r v a l b u m i n content i n 2 week n o r m a l ( N ) and dystrophic  muscle samples. PARVALBUMIN ps/meTSP N  EDL  n=5  DY  EDL  n=5  CONTENT  ue/me W E T WT  M E A N S.D. S.E.M.  19.74 7.74  M E A N S.D. S.E.M.  18.99 8.48 3.79  0.622  1.031 0.582 0.260  3.46  0.291 0.130  N SOL n=5  LESS  THAN  1.62  0.094  DY  LESS  THAN  2.33  0.003  SOL  Values reported for the mean T S P and W E T WT.  SOL  are  the  T S P = total soluble protein W E T W T = wet weight S.D. = standard deviation S . E . M . = s t a n d a r d error o f the m e a n  144  minimum  values  detected  DISCUSSION  This  work  represents  dystrophic  tissue  at  the  first  study  to  both  the  first  preclinical  compare  the  immunohistochemical  and  postclinical  changes  in  PV  study  stages  of  of  the  distribution  PV  in  disease.  and  content  murine  It in  is  also  fast  and  s l o w - t w i t c h skeletal muscles of dystrophic mice.  Immunohistochemistrv The  immunohistochemical  not  provide  does  however, twitch  new  consistent  muscle  information  with  and  distribution  the  regarding  proposed  correlates  data  well  role  with  of  of  PV  in  normal  the  function  PV  as a  histochemical  of  PV  soluble  (Ovalle  and  dystrophic  in  muscle.  relaxing  et  al.,  tissue  factor  1983),  It in  is, fast-  biochemical  ( J a s c h a n d M o a s e , 1 9 8 5 ) a n d p h y s i o l o g i c a l ( B r e s s l e r et a l . , 1 9 8 3 ) d a t a . Muscles concentration portion  of  of  in  distribution  of  tissue  the  soleus  and  light  1/slow-twitch comprised  fibers  probably type  parvalbumin  2  normal  fibers  fibers, a  and  on  a  of  respectively.  a  of  subgroup  1 fibers.  When  myosin  ATPase  reactions  myosin  population likely  muscle  intensities.  the  fiber  (Tables  145  that  numbers V  -  VII)  This  distribution  of  mice, and  the  The  50%)  type  distribution  of  was  found  in  the  lowest  in  2/fast-twitch as  and  intermediate  intermediately-stained  be  it  data  and  classified  are  present  (parvalbumin-positive)  These  cannot  a l . , 1982).  fibers  dark  cells  high  the  ATPase.  of  a  dystrophic  c o r r e s p o n d s to  fibers  type  genetically  (approximately  Those  of  investigate  parvalbumin-positive  staining  have  immunohistochemical  muscles  most  generally  1 9 8 2 ; H e i z m a n n et  to  the  definite  which  continuum  represent or  was  and  according  EDL  fibers  simultaneously  normal  percentage  There  (non-reactive)  Heizmann, to  typed  reports  highest  (8-10%).  attempt  fibers  the  fast-twitch  (Celio and  an  previous  from  of  a n d soleus o f  extrafusal  fibers  typical  was  EDL  The  sections  the  type  study  these  parvalbumin.  primarily  parvalbumin  the  parvalbumin  supports  composed  categorized compared is  evident  as  either  between that  not  the all  the  non-reactive  noted  by  fibers  Celio  exclusively  in  for  and the  parvalbumin  Heizmann  type  2  investigators  also  parvalbumin  immunoreactivity  typed  according  solely  muscle this  than  if  reported  to  typed  (1982)  fiber  that  are  and  continuum  described Pette, of  1986). type  not  tissue  fiber  experiences that in  since the  light  fibers  with  increased  C  fiber  type the  the  myosin  suggest  This  of  no  true  correlation  present  chain  study,  of  differentiation  the  composition  by  PV  than  between  the  PV  localization the  myosin  the  localized  intensity.  These  is  not  a  the  a  also  devoid  considered  Hence,  may  also  of  muscle  a  slower  results  true  of  correlation  types  and  In  fibers  chain  in  more  (Staron  ratios,  reflect  dystrophic  closely  was  with  of  a  muscle  also  staining  but  dystrophic  may  is  and  chains,  adult  It  complement  recently  varying  PV  that  ATPase  correlate  2A  the  progresses.  the  heavy  for  hypothesis  myosin  heavy  1 and  fast-myosin  stained the  reflect  co-existence,  2A  disease  may  was  actually  be  complement.  support  as  PV  type  intermediately would  was  activity.  between  chain  lack  there  noted  light  This  Therefore,  reactivity.  population  myosin  a  would  in  and  were  fibers.  noted  chains  population.  1  that  characterized  staining  fibers  type  ATPase  heavy  proportions  2A  variety.  parvalbumin  of  intensities  were  1 slow-myosin  correlate  the  greater  These  C  that  immunoreactivity  would  staining  heterogeneous  both  did  of  1/slow  degrees  between parvalbumin localization and myosin ATPase The  type  found  the  resembled  to  study  who  of  parvalbumin  immunohistochemical  the  displaying  some  according  of  possible observed  the  myosin  particular  fiber  types. Since always staining muscle  PV  a  pattern fiber  of  water-soluble A the  analysis samples  electrophoresis  addition,  a  possibility.  freeze-dried gel  is  redistribution  extrafusal  (Heizmann of  and  protein,  rat  two-dimensional  gels  of  may  fibers. et  muscle  revealed  artifactual  al., were  PV  to  be  This  responsible issue  1982). subjected be  heat-stable 146  redistribution  has  Typed, to  present extracts  during  for  been single  the  in  from  the  excluded muscle  type  is  checkerboard  two-dimensional  only  fixation  by  single  fibers  from  polyacrylamide 2  homogeneous  fibers. type  In  2B/FG  muscles  resulted  extracts.  in  These  very  high  concentrations  concentrations  correlated  of  PV  well  after  with  HPLC  the  analysis  known  of  contraction  the  same  and  half-  relaxation times. In the  the  present  distribution  of  immunostaining then,  the  mouse,  However,  thereafter,  PV  localization  was in  muscle  showed  a slightly  some  that  the  fibers  muscle than  would  appear  completed.  to  did  the  finding  in  enzyme profiles,  myosin,  in  r e s p o n s e to  al.,  1983a; G r e e n  to  follow  a  distinct  the  al.,  time  1984).  course,  2  weeks  muscle  2-week  muscles  a  slightly In  whereas  the  soleus  more  samples.  Therefore,  transition  of  the  stimulation Since  PV  the  and  in  and  not  still  the  the  rate  of  of  that  immunoreactivity  of  the  may  be  EDL  nonsoleus ATPase  exhibited  resembled  fast-  synthesis o f  have  PV  has  been  shown  that  those o b s e r v e d activity  various a  of  myosin  increased muscular  transitions  age.  mature  2-week  myosin  advance of  normal  with  percentage  closely  reports  the  obtaining  2-week  isoforms  S R occur in  age  contrast,  The  recent  Apparently  addition,  soleus.  with  muscles.  higher  In  in  parvalbumin  type are  factor  the  of  and  the  significant  normal  EDL  in  nerve  by  a  observed in  with  had  consistent  chronic  et  the  P V , and  the  not  P V - p o s i t i v e fibers.  week  is  was  counterpart.  of  both  before  changes  et  32  the  adult  in  attained  EDL  characteristics  plateau  This  week its  age  age i n  varies  that  percentage  muscle-fiber  twitch  (Klug  systems  useful  for  seem  indicator  of  maturity.  The similarity soleus  2  than  higher  been  evidence  was age-dependent  slow  32 w e e k s o f  almost  reactive  muscle  2 to  study,  obvious changes were  has  characteristics  more  from  No  profile  there  reactivity  parvalbumin.  pattern  adult  and  immunohistochemical  most in  dramatic  the  muscles.  preponderance muscles. muscles.  This It  localization The  of  trend  of  observed PV  toward  in  a  likely  was  also  that  noted  these  in  in  the  more  intermediately-stained  trend is  finding  this  adult even  distribution  in  the  myosin  147  of  dystrophic  fibers  "intermediate"  portion  both  the  ATPase  fibers  may  the  EDL was  fast  study with  brought and  staining represent  the  in  the  that about  slow profiles not  was  by  a  dystrophic of  these  only  type  2/FOG  fibers  classification "atypical",  but of  also  regenerating,  intermediate  "transitional",  fibers  or  degenerating, or may  "abnormal"  therefore by  immature  include  other  extrafusal  those  fibers  investigators  fibers.  This  referred  t o as  (Dribin  and  Simpson,  was  noted  1 9 7 7 ; O v a l l e et a l . , 1 9 8 3 ) . This  trend  muscles  of  of  was  PV  in  the  2-week-old  distribution  alteration  mice.  unaffected of  by  than  in  normal  at  this  distribution preclinical  and dystrophic  groups  myosin  ATPase  muscle; fetal  however,  myosin  1973)  in  it  back  soleus  However,  dystrophy  may  (Fitzsimons  dystrophic  regression and  reactivity,  disturb  muscle  not  the  immature  adopt  the  of  muscles.  The immunohistochemical  staining  d u e to  ascertain are  also  fetal-like  the  in  results  those  those that  muscle stain  the suggest  fibers  of  that  that  intermediately  the  fibers.  show It  have  been  shown  for  PV,  and  to  and Brooke,  the  or  a  muscles  be o f  contain been  mice. a  more  neonatal  a diminution  have  of  dystrophic E D L  from  would  of  presence  2-week-old  dystrophic  be  during  muscle maturation  profile  but, rather,  dystrophic  (Dubowitz  in  the  to  P V and  muscles m a y indicate  staining  also  development the  i n the adult  observed  dystrophic  P V localization  the i n c r e a s e d n u m b e r  whether  in  Although  to  the  it  appears  the n o r m a l  complete  but  respect  the  was  is n o t a f f e c t e d  with  fibers  2C in  pattern  differences  their  least  state, P V d i s t r i b u t i o n  P V i n the adult  state  more  At  process.  a failure  staining  immature  become  maturation  weeks,  2  in  localization  only  immunostaining  does n o t s e e m to alter  m a y indicate  the concentration despite  dystrophy.  a n d H o h , 1983) a n d type  to a m o r e  does  muscular  PV  the  Not  at  b y 2 w e e k s o f age i n the m o u s e , a n d its d i s t r i b u t i o n murine  Thus,  stage.  established  stages o f  muscles.  not  variations,  that  incipient  adult  slight  proportions  resembled  the  the  Other  PV-specific  dystrophy  P V similar of  of  do  in P V  interest  fetal  not  to  myosin  described  as  "transitional" or "abnormal" b y other investigators.  i) M u s c l e S p i n d l e s Because  serial  sections  along  the  lengths 148  of  individual  muscle  spindles  were  not  performed PV  in  in  the  present  study,  very  intrafusal  muscle  fibers.  It  distribution reactivity muscle  of as  PV  muscles staining  in  sections  stained  for  both  normal  of  intrafusal  discussed  spindles  positively  in  of  the  intrafusal  zones  the  apparatus  spindle if  it  PV  may  is  a  On  not  truly  of  be  of  an  cytoplasmic  that  other  those of  were  noted  in  intrafusal PV  is  no  the  bag  age  affinity  polar  fibers in  in  in  the  that  of were  2-week  regions.  No  equatorial Since  polar  intracellular  and  most  of  of  the  region  here.  of  ATPase  the  polar  the  in  regions  fibers  concentrated  for  exist  groups.  within  also be  localization  myosin  in  observed  found  would  with  of  in  chain  immunoreactive  either  fiber  and  was  the  differences  genotype,  chain  however,  protein  regional  to  hand,  mice  that  about  occasionally contained  spindles  surprising  concluded  Regardless  fibers,  muscle  be  similar  muscles the  can  likely  1.  and dystrophic  juxtaequatorial contractile  adult  PV.  is  fibers,  Chapter  of  little  However,  structures,  this  result m a y represent evidence f o r some particular intracellular localization. PV spindles and  immunoreactivity in  adult  Heizmann, in  mammalian  chain  1984)  to  (Celio  PV  shows  EDL  spindles  of  fibers  the  this  appear  chain fibers  that  separately  as  neither a  means  muscles that  presence may the  faster  account  1982),  it  for  is o f  not  of  those  the  in  the  of  interest  that  nor  myosin  intrafusal  fiber  It  bag  one  appeared the  bag  types  muscle  fibers  (Celio  known  that  fibers  (Boyd  and  of of  the the  chain chain  identical  in  can  there  is  fiber fibers  the  f i b e r s , it  staining since  was  is  extrafusal  ATPase  of  immunostaining  reaction  only  As in  fibers  EDL.  than  positive  1.  the  the  properties  reactivity  chain nuclear  that  the  Chapter  determining  two would  be  used  no  true  two. PV  sign  of  immaturity  synthesis  of  PV  a  in  ATPase  of  be  not  immunoreactivity of  one  reported  relaxation  as d e s c r i b e d i n  c o r r e l a t i o n b e t w e e n the The  investigators  Myosin  PV  in  soleus muscles but  may  Heizmann,  reported  soleus but  have  Although and  been  These  immunoreactivity.  intrafusal  fact  and  1982).  unequivocal  Smith,  rat  has  in  nuclear  bag in  generally  fibers  the  synthesis  proceeds 149  of  that  muscle of of  this  spindles protein.  myosin  in  the  2-week  However,  isoforms,  and  the that  adult  myosin  intrafusal fibers fast  ATPase  fibers  is  a  bag  to  this  1),  fibers  it  to  is  and  PV  that  not  age  in  been  have  and  It  also  of  fibers  contraction  may  weeks  reactivity  rapid  1984).  2  PV-positive  intrafusal  fibers  was  by  Bag2  Smith,  intrafusal  Why  established unlikely  their  (Boyd  of  study.  are  phenomenon. due  properties  in  patterns  Chapter  fibers  bagl  physiological observed  (see  developmental  nuclear  compared  staining  is  correlate  observed in  bag  procedures  may  fibers  bag  times  that  the  of  described  relaxation  possible  murine  as  when  the  unique  immunolabelling  of  the  adult  muscles  is u n k n o w n .  Parvalbumin Content Although a  particular  cellular  PV  cell,  content  exercise. in  immunocytochemical they of  The  this  age  the  dystrophic  thereby  not  provide  protein  by  immunohistochemical  distribution  and  do  staining  not  groups.  only  More  determine  the  between  It  whether  quantitative  intensity  results  noteworthy  samples.  very  in  muscles, were  the of  of  information. the  this  study  but  also  therefore  interest  the  immunohistochemical  stain  between in to  a  very  in  these  could  the  differences fiber  localization  quantify  PV  subjective  dramatic  individual  the  results  localize  Determining  is  revealed  alterations  was  specifically  types  of  PV  in  changes  be  and  substantiated  biochemically. The  biochemical  immunohistochemical electrophoresis consistently for the  the  greater in  the  to  be  more  in  dystrophic the  EDL  concentration  adult than of  PV  presence  and  prevalent  support  the in  PV  the  age  the  group.  than  in  soleus  between  Parvalbumin the  adult  of  the  fast-twitch  and 150  results  PV,  content than  in the  values  were  and  as  of  the  animals.  preceding  determined by  soleus.  normal  RIA This  approximately  This muscles  by  gel were  is  true  population, and  approximately  slow-twitch  the  determined  age i n  soleus, younger  the  of  EDL  b o t h 2 a n d 32 w e e k s o f  EDL in  here  The  immunoblotting,  t i s s u e s a m p l e d at  2-week  presented  experiments.  and  shown  findings  12  times  times  difference is  27  in  greater in  consistent  the with  similar  trends  Heizmann,  1984;  hypothesis muscles  reported  that  previously  Heizmann  PV  et  content  is  in  al.,  type  of  species  1982).  fiber  composed predominantly  other  Collectively,  specific  type  (Leberer  2  (Celio  fibers  these  and  have  a  on  the  and  Pette,  results  support  Heizmann,  higher  PV  1986a,b;  1982),  the  in  that  concentration  than  muscles that are p r i m a r i l y type 1 i n c o m p o s i t i o n . The optimal the  sensitivity  dilution  iodination  approach small  The  is a n  1986b). low  of  titre  presence samples  of  of of  one  in  source  various  s u c h that  by  the  the  a  The  observed is  in et  the  antibodies  of  in  the  T S P values are  same  as  1982;  the  is  the  that  the  antisera  serum.  fact  that  may  have  than  ours  showed  that  the  and  the  result  of  a  antigen,  or  a  1985;  the  1979).  compared Endo  et  antiserum  been  T P over  to al.,  had  masked  a  by  the  treats  his  T S P values  are  Heizmann  the  for  immunochemical  low  al.,  used  (Cooper,  are  et  antiserum,  antigen  standard  study  well,  the  be  determinant  As  lower  may  the  Klug  the  of  of  competition  present  al.,  the  purity  antigenic  discrepancy  affinity  the  specificity  remembered  similar  RIA  this  and  be  identity  (Heizmann for  other  should  has  the  depends  antigen,  which  whose  reports  1:400-500.  It  that  obtained  possible  and  1979).  material  published  A  antibody  competitor  values  previously  of  indirect  quantity PV  ratios  radioimmunoassay  (Cooper,  amount  larger  of  heat  greater than ours. The 2-week  immunohistochemical  normals  concentration PV  content  between pattern  of  of is  age  was PV  in  70%  of  groups  PV  not  data  significantly the  E D L , however,  that  were  distribution  different  found  in  virtually has  to  established  that  distribution  of  the  dependent.  normal  identical  been  from  is a g e  the  overall  adult  each by  2  adult  By  2  EDL.  other.  of  weeks  Soleus  of  age,  in  muscles.  Therefore,  weeks  PV  the  of  the The  age  the  concentrations although  the  synthesis  of  P V w i t h i n t h e p o s i t i v e l y - s t a i n e d f i b e r s has n o t r e a c h e d its p e a k . The have 1982;  present  detected  PV  Berchtold  findings by  and  are  postnatal Means,  in  agreement  day 4 or  1985;  5 in  Leberer 151  with the  and  previous  E D L of Pette,  developmental  the  1986b).  rat In  (Celio and  studies  that  Heizmann,  one-week-old  mice,  the  PV  content  initial  represents  synthesis  asynchronously al.,  1979).  and  in  5-13%  time  various  While  of  that  course  of  fast-twitch  mature  levels o f  found the  muscles  PV  in  have  the  adult  increases (Leberer  been  (Klug  in  and  noted  et  PV  the  1985).  appear  Pette,  in  al.,  1986b;  The  to  Le  occur  Peuch  gastrocnemius  and  et  psoas  J  of  the  after in  rabbit 17  the  1985;  by  weeks EDL  subsequently  that  the  et  gastrocnemius  after  and  al.,  increased  15  Means,  synthesis  by  its  maximal  levels  have  1985). PV  followed  In  to  PV  day  more was  20-fold In  7  (Le  20  at  to  1982,  Jasch  5  days  with  developmental  regulation  the  differentiation  processes  rapidly-relaxing  extrafusal  These  a  include  synthesis myosin by  of  a study  migration isolated molecular  chain  and  the  cDNA  to  The  It  was  of  these  lead  relaxation calcium  to  time  cycle,  various  the  after  et  PV  involved  is  early  embryo a  PV  that  cells that  al.,  PV  from  the  role  of  the  development of  tadpoles,  and  its of  active  or  and  mRNA  et  PV  occurred  in  expression  expression  al.,  absent at  of  correlate and 1984).  and  the  pump  and  been  supported  differentiation,  available is  studies,  to  calcium  tissue  (Kay  leg,  reaching  these  tetanus,  recently  in  the  Heizmann,  SR  has  shown  fast-contracting  the  This  levels  regulated.  1985;  twitch  particular  coordinated 152  a  rat and  have  appears  of  Means,  1979).  appearance were  and  the  hatching,  muscles  formation  after  in  Peuch  demonstrable  suggested  in  fibers, (Berchtold  found  muscle  PV  which  encoding  was  first  when  (Le  see i f  events  it  in the  segments  probes  differentiation  of  kinase  designed  development.  apparatus.  decrease  elements  light  muscle  of  of  of  PV  Moase,  maximum  t h e r e f o r e , i n d i c a t e t h a t P V s y n t h e s i s i n s k e l e t a l m u s c l e s is d e v e l o p m e n t a l l y The  and  muscles  Results  of  postnatally,  its  in  obtained  levels  investigators  hatching  1979).  been  study  day  other  4-5  not  age, adult  achieve  embryo,  al.,  of  al.,  detected  muscle  et  et  have  developmental  before  pectoralis  EDL  weeks  (Jasch  day  days  Peuch  the  8-12  first  chick  2  in  recent  by  the  the  By  achieved  a  begins  in  levels  1986b).  been  1985).  of  similar  mRNA  synthesis by  birth,  Pette,  mouse  muscle,  and  active  weeks  (Leberer  of  Klug  (Berchtold  9  the  1987).  cell Using  antibodies during  as early  time  of  somite  of  the  contractile  PV  and  contractile  -  proteins  also  supports  the  belief  that  PV  plays  a  role  present  study  (Table  in  the  contraction-relaxation  events o f skeletal muscle. The  biochemical  dystrophic  EDL  the  observed  was  noted  decrease weeks  of  the  PV  data  postnatal  stages  of  IEF  age  in  present  32  the  significantly  week  reported  age ( K l u g  published  these  contains  in  immunohistochemical  in  in  data  on  et  previously  a l . , 1985).  the  in  disease  protein (Jasch  there  the  EDL  the  dystrophic  probably  due  is  to  of  et a  This  al.,  reduction  findings in  muscle  Normalized of  stage  PV the  of  the  than  the  40% at  15  12  with  progressive  from  scans  to  drop  disease  values  consistent  data  81%  with  muscles  during  from  adult  normal  larger  are, however,  Therefore,  advanced  from  is  the  consistent  dystrophic  mouse  depletion  that  EDL,  diminution  1982).  gradual  showed  normal  fast-twitch  these  mouse.  the  the  81%  distributions  that  is  An  various  Both  indicate  study  than  EDL.  for  gels  of  PV  changes.  dystrophic  changes  of  less  VIII)  32  weeks  noted  resulting  of  in  the  from  a  d e g r a d a t i o n o f P V as t h e 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 y p e s c h a n g e .  fact,  On  the  the  concentration  normal  s o l e u s to  support  our  muscles  of  are  other  the  on  a similar  of  levels  data  becoming  hand,  in  approaching  the  alike  the  was  adult  those  of  animals. with  It  respect  not  the  dystrophic  appear,  content  and  in  the  soleus  distribution  would to  observed  dystrophic  immunohistochemical  dystrophic  more  PV  trend  soleus muscles.  surpassed  EDL.  that  Hence,  of  PV  in  then,  that  the  distribution  the  of  of  these  fast  EDL  In the  results  and and  slow soleus  P V , as w e l l  as  in  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 t h e g e l s . The  results  noteworthy.  from  Parvalbumin  the  immunoblots,  the  immunohistochemical  fiber  population  explanation whole  our  for  muscle  and  in this  was  only  the may  experiments undetectable  minimal  data  values  indicated  normal lie  in  homogenates  on  soleus  the  fact  and  the  by  could  that,  gel be  taken  contained that not 153  all  the  from  normal  soleus  muscles  were  barely  evident  electrophoresis, obtained  with  the  collectively,  22%  some  of  level  biochemical single  RIA. of  data  fibers,  extrafusal A  was  on  However,  the  PV.  also  possible  collected  whereas  on the  immunohistochemical addition,  even  indicating  the  staining  though  identified  there  presence  of  is  PV,  PV  a  the  in  sections  relatively actual  of  high  PV  individual  muscle  percentage  concentration  of  cells.  positive  within  these  In fibers  fibers  may  n o t b e i n s u f f i c i e n t a m o u n t s to b e 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  during  obvious  the  preclinical  noteworthy  feature  values  for  in  adult  the  for  the  EDL  PV  in  the  the  two  muscles  does  disease a  EDL  diminished  the  the  dystrophy  when  increased  that  the  of  dystrophic  have  support  alteration  stages  has  resembles  muscular  emerges  the  suggest  closely in  both  soleus  findings  that  in  not the  comparison  and  that  of  above  muscles  adapting  hypothesis  that  differentiation  the  the  are  those  stages  dystrophy  may  with  (see  made.  of  to  concentration  and  The  the  intermediate  of  dystrophy. an  to  calcium  the  A  of  the  form  PV  values  muscle.  These  that  These  immature  PV  postclinical  content  2-week  an  of  IX).  E D L , whereas  reflect  respect  Table  preclinical  2-week  preclinical  process  the  mouse  2 J  soleus m u s c l e s are  beyond  the  dy  of  significantly  in  affect  more  alterations  state  regulatory  or  an  system  ( K l u g et a l . , 1 9 8 5 ; P e t t e et a l . , 1 9 8 5 ) . As altered  illustrated by  16-20  K D  dystrophic Some,  or  binding quite  18  murine  34,  dystrophy.  range.  Those  fast  or  of  higher  they  was  not  particular  characteristic  reciprocal  calcium-regulated that  Of  A  these  PV  bands  muscle.  probable  K D  Figure  murine  all,  LC2-fast,  in  the  interest of  proteins. the  the  a  slow  to  the  myosin  muscle  muscles in  may  and  calmodulin,  dystrophy  may  noteworthy  that  not  (Fitzsimons  and  Hoh,  interrelationship  is  all  16.7  also  reflect  the  a  myosin  1983;  between  KD.  Jasch  these  An  alteration  loss  of  LC's and  proteins  the  are  154  the  normal  similarly  Moase, and  in  1985).  PV.  in  the  now  appear  in  the  dystrophic be  of  18 K D ; L C 3 - f a s t , 16.5 K D ; L C I - s l o w , 2 7 K D ; L C 2 - s l o w ,  these  19 K D ) , LC  differentiation affected It  Further  is  either  (LCI-fast,  myosin  was  bands  also  chains  that  of  the  position  light  protein  number  occurs  proteins  According various  soluble are  phenomenon  molecular-weight  represent  only  soleus. calcium-  bands  it  22.5  is  KD;  troponin-C,  composition  in  process.  is  It  in  murine  dystrophy  not  known  what  speculation  should  not  the be  made  until  present  the  identity  study  proteins,  would  namely  of  be  these to  calmodulin  proteins  determine  and  is  known.  whether  troponin-C,  are  A  critical  the  other  affected  in  follow-up  to  the  major  calcium-binding  similar  fashion  a  by  the  dystrophic process. Besides  the  various  physiological  Ca  muscle  (Walsh  in  types  restoring  not  Ca  stimulate  1984)  2 +  Ca  -dependent  2 +  Ca  involved  in  calmodulin function  2 +  -uptake  could  regulation  to  in  be  -transport  activator  1980),  SR  role  remains  of  to  Ca -release  PV  is a l t e r e d  in  in  neutral  protease  preservation hamsters of  of  exhibit  a  (Sugita no  dystrophic  accumulation  chicken in et  observed  this  SR of  phase  this  of  three  troponin-C  it  may  is  to  and  muscle be  possible  cycle.  also contribute  al.,  (Chiesi  protein  the  does et  skeletal  However,  pretreated and  1980).  no  dystrophic  with  If  the  the  changes  of  apparent  the  the  a  relative  muscle  calmodulin  correlation  and  with  skeletal  between  muscle  calcium-activated  troponin-C  Studies  differences  therefore, in  for  all  the  dystrophy.  troponin-I al.,  that  in  (Eibschutz  skeletal  appears  may  muscle  detectable  animals;  it  as  calmodulin  muscle  regulation  relaxation  dystrophy  decrease  demonstrated  and  calcium  dystrophic  troponin-T  have  normal  the  from  slow  the  kinase  although  from  in  such  substitute  contraction.  the  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 Extracts  or  2 +  calmodulin  also  for  fast  muscle  chain  And,  in  of  substitute  ATPase.  determined,  calmodulin,  effectively  either  of be  can  of  light  of  calcium-regulation  calmodulin  myosin  actomyosin  the  the  activities  calmodulin  involved  Although  the  of  al.,  may  1982).  and  et  -sensitivity  calmodulin  Carafoli, SR  2 +  enzyme  exists  recorded  from content  between  changes  in  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 . , 1 9 8 3 ) . Although increased close  the  decrease i n  of  protein  rate  similarities  in  affected  differently  observed  in  PV  muscle  proteins  in  degradation  (Garber  et  proteins by  content  the are  such  as c a l m o d u l i n ,  same largely  disease. due  to 155  dystrophy  1980),  troponin-C In  a  al.,  is  murine  conversion  largely it  and  appears  that,  P V , each  one  dystrophy, in  the  attributed  to  an  despite may  be  the  alterations  phenotypic  expression  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  GENERAL  It  was  intrafusal  these  sensory of also  is  and  in  muscle  tempting  spindles to  and  nerve  supply  speculate  different  which  spared  the  dystrophic  of  various  myosin  may  fact  fibers  in  in  the  one  considers which  the  is  its  intrafusal  fiber  those  its  gene heavy  chains  any  It  is  own  the  mice.  been  be  innervation  described  possible  by  As  actual  myosin  histochemical  pattern both  light  complexity  which  muscle's  state  It  fibers,  is  and  combination  chains  changes  the  neurotrophic  extrafusal  and  this  mechanism.  neighbouring the  in  that  governed  well,  neonatal  However,  safeguard  may  expression.  in  complex  have  innervation  of  observed  dystrophic  muscle.  from  mask  were  dystrophic  the  are  intrafusal  to  that  alterations  alterations  polyneuronal  which  composition  when  the  influences are  no  spindles  interesting  receptors  motor  that  muscle  itself  sensory and  the  unexpected  fibers  observation to  not  DISCUSSION  or  within  may  the  occur  as  a  result o f the disease. Although perhaps  a  PV  may  marker  localization  could  for not  rigorous  investigation  of  intrafusal  the  be  a  useful  early be  neuromuscular  detected  into  the  fibers,  indicator  in  the  actual  nothing  of  a  disturbances,  intrafusal  distribution  conclusive  significant  fibers.  of  can  PV be  of  changes  However, throughout  said  maturity,  about  in  without the  its  and  a  entire  PV more  length  appearance  in  a  decrease  in  whereas  an  d y s t r o p h y w i t h r e g a r d s to t h e s p i n d l e . The  data  presented  parvalbumin  content  increase  parvalbumin  in  alterations muscles for  the  are  in  occurs  half-relaxation role  Chapter  in  the  content  consistent  proposed  in  of  with  2  of  this  extrafusal occurs  the  times.  fibers  in  the  known  This  parvalbumin  of  a  indicates  the  also  that  soleus occur  provides  soluble  that  dystrophic  dystrophic  changes  study as  thesis  with in  further  relaxation  EDL  age.  the indirect  factor  in  These  fast-twitch evidence fast-twitch  skeletal muscle. In  both  the  dystrophic  EDL  and  soleus  157  there  appears  to  be  a  conversion  of  extrafusal  fiber  types  matched  muscles.  neonatal  muscles  parvalbumin. does,  increase  in  at  the  differentiation  fiber  types  with  the  in  to  that  the  the  to  the  SR  both  immature in  and  maturation  or  f i b e r s , p a r t i c u l a r l y the elements i n v o l v e d i n the C a There  are  in  murine  altered  several  withdrawal  of  myosin,  an  increase  the  for  1983;  SR  correspond and  and  Means,  their slow  normal and  ,  differentiation  innervation,  the  glycolytic  and  a  decrease  in  contraction  synthesis  of of  Analysis  fast-twitch has  counterparts.  intermediate  murine  myosin,  alteration  dystrophic  and  a  and  alters  fibers.  The  distribution is  of  consistent and  Ca  2 +  Muscular  dystrophy,  of  in  fast  off  normal  are  fast  muscle  and  muscle  is  the  the  or  includes  the  and  of  characterized of  times the  Ca  1982;  myosin  and  components  muscle  uptake-capacity  Heizmann,  proportion  prolonged  slow-twitch  part  (Fitzsimons  these  is  muscle  of  and  isoenzyme  muscle  decrease i n 158  way  maturation  half-relaxation  that  (Celio  myosin  dystrophic  an  The  native  of  enzymes  elements  parvalbumin  of  shown  these  in  parvalbumin  maturation  switching  of  Synthesis  observed  This  entities.  muscle  activity  the  1985)  skeletal  Normal  higher  cycle.  2 +  the  1985). in  Moase,  2 +  that  muscles  of  some  the  of  slight  isomyosins  differentiation  in  1985).  with  distribution  dystrophy.  polyneuronal  Ca  Dhoot,  indications  in  muscle.  Interestingly,  calcium-regulated  those  and  types,  resemble  The  appearance  regenerating  content  fiber  development.  the  of  dystrophic  extrafusal  other  influence  of  the  age-  distribution  muscle.  dystrophy  maturation  normal  dystrophic  resembling  that  parvalbumin  state  the  these  and  EDL  hypothesis  or  in  in  completely  immature  soleus  dystrophic  subsequent  of  more  seen  not  content  found  in  that  does  the  dystrophic  support  stage  either  parvalbumin  the  than  myofiber  reported  in  conversions  the  appears  of  form  of  in  adult  and  an  neonatal  of  age  disease i n  reflect  a  form  levels  bands  this  reported  metabolism  in  process  may  of  the  m u s c l e , also  observed  intermediate  concentration  protein  slow-twitch  therefore,  weeks  parvalbumin  the  parallels  2  more  intermediate  resemble  molecular-weight  changes  a  This  The  however,  to  Hoh,  when by  of  (Vrbova, 2 +  cycle,  Berchtold  light  chain  1983;  Jasch  compared  to  increase  in  an  myosin  light  chain  3  (fast)  and  myosin  investigated  the  alterations  in  However, 1  a  fibers  the  the more  of  2  myosin  light  type  mobility  or  (fast-phosphorylated).  chains of  patients  LC3  was  than  noted  in  in  of  isomyosins, Fitzsimons and  muscle  from  reflects  muscle  affected  with  Duchenne  or  and  control  dystrophy  was  compared  et  type  fibers.  large  al.,  Thies  and  to  (1979)  found  normal  by  1982). In  the  of  presence of  these  In  muscle.  fetal  type  human  myosin  this  myosin  workers  in  of  fetal of  no  particular,  a study  amounts  percentage  estimated  muscle  (Takagi  that  The  and  c h a i n s has b e e n d e t e c t e d  found  suggested  immaturity.  human  light  muscle  (1981)  Samaha  L C 2 when  these s l o w  Hoh  patients \ and  regeneration  dystrophic  myosin  55% of  skeletal  Duchenne  in LCI  diverse composition  D M D  fast-type  light-chain  in  isoform  in  at  1-12%  1986)  also  muscle of  the  total amount of m y o s i n . A the  more  recent  existence  of  myosin  human  dystrophic  stained  positively  fibers  to  be  muscle  are  cycles  of  normally the  immunofluorescence  muscle. for  confined necrosis  20% to  and  accompanied  of  isoenzyme  patterns  immature  normal and  many  muscle proteins  with  presence the  of  In  and  in  fetal  and  of  to  recently  dystrophic  characteristics.  159  fiber  changes  may  in  of  reactive  in  dystrophic  from  successive  be  that  is  delayed  in  fast-twitch  similarities  in  mouse  (Petell  et  accelerated  et a l . , 1 9 8 0 )  abnormalities  just  mentioned,  with  hindlimb  enzyme  and  muscle  and  al.,  1984;  degradation  may  These  in that  demonstrated  dystrophic  and  fibers  maturation  also  demonstrated  muscle (Garber  histochemical  resulting  myosin  synthesis  myosin  reported  myosin  extrafusal  synthesis  adult  fetal  percentage  fibers  findings,  both  from  fetal  (1983)  slow  been  increased  that  myosin  these in  the  muscle  Desypris  metabolism  muscle  formed  Alternatively, of  al.,  small  estimated  newly  addition  An  many  suggested  presence  have  1984).  immature  contractile  the  anaerobic  muscle  Pette,  of  cessation Parry  mice.  Reichmann  the  the  and  They  et  indistinguishable  observed  myosin,  population  methods  of  workers  41%.  (Schiaffino  were  regeneration. by  dystrophic  other  and  process.  immunohistochemical muscles  a  that  These  anti-fetal  between  dystrophic  isoforms  study  account  correlate indicating  of for well a  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 s t a t e . For to  a  include  from  more in  this  another  would  have  revealed Due  complete study  provided  the  tissue  developmental data  transformations  to  study  difficulties  in  this  maturation  samples  stage, on  in  of  a  PV  from  such  more  as  obtaining  8-12  weeks stage  during  adequate  it  1-week-old  immature  distribution  issue,  a  would  dystrophic of  of  age.  of  young  been  mice  those  age  and  stage  useful  and  These  development  transitional  numbers  have  of  groups  may  have  8-12  dystrophic  weeks.  animals,  and  i n the t i m e constraints, s u c h a n extensive s t u d y was not u n d e r t a k e n . Why  is  concomitant and  what  with are  parvalbumin  skeletal  has  electrical  a  consequence  of  the  depletion  of  localization  of  the  in  earliest  content  dystrophic in  and  developing  and murine  most  also  muscle  fiber  transitions  are  type  less but low  fast-twitch  et  markers  in  more  secondary of  a  transformations  observed  expression  to  fibers  in  et  is  the  display  (Klug  it  in  but  2/FOG  content  fiber  been  muscle  phenotypic  its  soleus,  contain  that  sensitive  denervation,  the  that  a l . , 1984; M u n t e n e r  skeletal  has  and  muscle  decrease protein  type  fibers  induced et  A the  1/SO or  EDL  slow-twitch  fibers  degenerating  experimentally  the  of  muscle  a l . , 1984; S t u h l f a u t h  one  conversion  a  in  alterations?  necessarily type  cross-reinnervation  in a  to  parvalbumin  stimulation,  to  or  fast-twitch  protein  these  Parvalbumin  conversions  altered  observed  as  dystrophic  of  population  are not  rapidly  a l . , 1 9 8 3 a ; G r e e n et  an  changes  to  the  related  regenerating  respond  established  in  calcium-binding  merely  types  atypical,  fast-to-slow  Because  not  PV  implications  immunoreactivity.  been  induced  is  in this  functional  These myofibers  muscles et  reduction  increase in  fiber  either  parvalbumin  It  an  the  in  parvalbumin. perhaps  a  content  conversion  (Klug  there  al.,  1985).  of  activity-  al.,  1983a,b).  response  likely  to  that  effects  extrafusal  the  and  a  myofiber  types. We as  myosin  at  the  cannot  speak  ATPase  level  of  fiber  specific  of  muscle  type  or  systems,  PV not  content. merely 160  based on  The as  a  one  parameter  transformation single  entity  must with  be  alone,  such  considered  respect  to  the  muscle  fiber.  enzyme  profiles,  These  It  it  similar  follow  would  observed  been  an  appear  modifications to  observed  that  sarcoplasmic reticulum,  alterations  Thus,  has  the  ordered  that  of  plasticity  myosin  time  murine  the  fiber  type  isoforms,  course  as  dystrophy  various  in  and  they  systems  response  to  (Green  changes et  al.,  developmentally  in  of  involve  PV  are  induces,  functional  described  transitions  a  the  other  graded  1984).  regulated.  fashion,  extrafusal  in  muscle  neuromuscular  the fiber  conditions  (Pette, 1980). The present  changes  study,  and  decreased calcium fibers  have  a  acquires  uptake  ability  and  in  peak 2 J  the  turn  delayed  well  that  are  with  muscle the  asynchronous  changes  in  and in  PV  the  the  or  al.,  been  1  attributed 1983).  content.  it  is  in  to  presence o f  the  a  to  An tissue,  time-toin  the  1983).  slow  also  of  The  myosin  induce  consequence  fast  metabolism.  process  changes  appears  2  calcium  and  Desypris,  these  the  adipose  times  and  in  types.  calcium  dystrophic  the  dystrophic the  fiber  Parry  as  As  fibers,  in  Type  possible  overall  the  role  PV.  fibers.  half-relaxation  dystrophy  process  in  muscle  However,  Thus  differentiation  slow  observed  crucial  expression  of  1983;  a  slow  muscle's  prolonged  et  play  distribution,  the  features  muscles  alterations  phenotypic  the  Desypris,  fact  type  type  affect  in  have  than  intermediate  (Bressler  reactions  decreases  to  characteristic  mouse  (Parry  addition  the  naturally  reflected  dystrophic in  fiber  of  the  may  SR  mirrors  would be  physiological  fast-twitch  intermediate  amounts  would  of  S R , in  developed  tissue  tissue  strain  2 J  this  in  reports,  the  highly  relative  tension  /dy  of  more  of  distribution  previous  uptake  a  connective  This  dy  in  fiber  in  more  muscle  increase  in  in  correlate numerous  an  unknown  genetic defect. Although  the  changes  prolonged  half-relaxation  muscle  not  (Bressler  is  et  reinnervation  paralleled al., study  PV  time by  1983). of  in  of  a  this  and  similar slow  in  the  muscle,  decrease i n  A fast  content  its  an  increased  half-relaxation  finding muscle 161  dystrophic  was of  the  fast PV time  observed rat  muscle content  mirrors in  the  by  32  weeks  in  a  recent  (Miintener  et  al.,  the slow  of  age  cross1986).  Cross-reinnervated physiological content  the  30-80  muscle  face  of  1986).  muscle  characteristics  was  relaxing  fast  times  may  a  greater  more  transformations.  as  These  the  the  PV  its  suggest  slow  of  than  to  disturbances  a  those  contain  might  neuromuscular  to  disturbance  This  with  normal  slow  than  normal  that  PV  well  as  had slow  fast  Thus  an  useful  indicator  also  a  of  the  However,  PV  the  contracting/slow muscle  (Miintener  for  early  abnormal  support  and  relaxing  interaction marker  histochemical  slow  contracting/fast  nerve-muscle a  similar muscle.  muscle.  a  is  observations  nerve  et  al.,  changes  changes  hypothesis  in  in  in  muscle  that  murine  dystrophy m a y suffer f r o m an altered nerve-muscle interaction. At  the  dysfunctional seen  in  PV  alteration light  of  present  time  it  is  in  way  in  the  any  localization  in  the  this,  it  and  antigenicity might  be  not  clear  dystrophic  content of  the  useful  whether  in  to  tissue.  the  protein run  the It  present due  an  to  amino  different is  study  possible  that  may  the  chemical or acid  muscle  be  and  the  are  changes  result  structural  analysis  proteins  of  an  changes.  In  HPLC  profile  on  P V extracted f r o m typed single fibers o f dystrophic muscle. It  is  difficult  concentration Certainly muscle turn  a  may  have  and  lead  coworkers  determine on  diminished  fiber,  can  to  protein  perhaps  to  the  have  sarcoplasmic  Ca  -binding  whether  levels  to  be  the  responsible  muscles. calcium an  In  the  for  impaired  to  an  capacity  in  the  functioning  of  state, in  360  alter of  SR  to: 2 +  Ca  Ca 2 +  (1)  -uptake  decrease i n P V . 162  of  the of  -buffering  this  muscle.  capacity  of  are  it  is  in  the  not  the  rioted  known reduced in  these  sarcoplasmic  membrane  in and  significantly  sequestering  the  Klug  reduction  concentrations  altered  skeletal  disease.  However,  that  parvalbumin  proteases, w h i c h  muscle  plausible  and  2 +  muscle  calcium  an  system  of  tissue.  seems  in  -dependent  typical  fast-twitch  now  Ca  the  pmole/kg  intracellular it  alteration  -buffering  dystrophic  response  the  2 +  an  destruction  dystrophic  increased  rises  would  cell a  in  Ca  activation  estimated  dystrophic  concentration  content  lead  observed  effect  intracellular  irreversible  (1985) 2 +  the  what  free  permeability,  system,  and  (3)  (2) a  A  decrease  prolonged  in  as that  well the  suggested  that  as  those  of  is  under  PV  are  muscle  1986b).  Denervation  of  that  of  concentrations.  This  higher  of  turnover  These  findings  important of  types.  adult  in  fast  turn  in  that The  the may  in  mouse  "arrested  development  fast-twitch which  in  rate  affected  in  exhibits PV  a  observed may  in  evidenced  a failure  of  the  a  It  normal  nerve-  has  been  activity  by  since  denervation, and  Pette, PV  suppression  in  PV  attributed  to  the  and  Pette,  muscle  loss o f a  a  in  dystrophic  by  muscles,  decay  was  (Leberer  a  rat on  rapid  delayed  reflect  righting  (Leberer  in  a  of  1986).  muscle  reduction  muscle  of  motor-neuron  results  muscles,  to  1985,  denervation  muscle  of  PV  al.,  with  neuromuscular  dependent  fast-twitch  by  developing  alterations  is  fast-type  presumptive  changes  be due  by  et  several  denervation  muscle  Miintener  muscle  the  and  skeletal  unaffected  the  concomitant  observed  regulation  the  fibers,  been  fast  1984;  appears  proteins  implications.  This  al.,  difference  of  in  developing  suggest  differentiation  PV  et  extrafusal  cross-reinnervation  suppressed in  slow-twitch  to  on of  also  mutant  neural  whereas  compared  PV-positive  have  the  (Stuhlfauth  PV  concentrations  on  expression  interaction  of  time,  Studies  response"  muscle  number  half-relaxation  disturbances.  indicate  the  the  1986a).  may  have  acquired  transformation  in  state fiber  nerve-muscle interaction  in  p o s t n a t a l d e v e l o p m e n t as s u g g e s t e d b y J a s c h a n d M o a s e ( 1 9 8 5 ) . In and  a study  coworkers  inspite  of  prevent  the  (1983)  complete  degenerate  It in  hand,  influenced  neurotrophic  nerve  dystrophic  reinnervation  by  a  Muscle and  of  the  murine  expression  influences  that  of  appeared  other.  was  trophic found  degeneration  observed.  each  of  that  dystrophic both  dystrophy  by  nerve  and  degeneration  abnormal  influences  the  was  dystrophic in  the  be  fibers and  No  do  to  163  of  the  the  light  altered  not were  programmed  to  independent  of  dystrophic  degeneration, In  could  influences  extent  Saito  degeneration  muscle  genetically some  Nerve  mouse  2 J  underwent  effects  for  dy  myotrophic  expression alone. responsible  the  normal  were  so  additive  influences.  gene  part,  nerve,  muscle  could  muscle in  muscle  nerve. and  both  neurotrophic  may,  normal  and  of PV  on this,  the  gene other  abnormal  expression  in  dystrophic  t i s s u e , as  well  as t h e  changes i n  the  SR  as s e c o n d a r y f e a t u r e s g o v e r n e d b y t h e u n k n o w n p r i m a r y Although tissue in  is  flow.  function  rapid  physiological role  believed  axonal  may  the  to  to  function  The  author  shorten  succession.  It  the  was  in of  of the  a  refractory  also  within  the  and  activity  within  nervous  processes a n d  regulation  of  concentration  various of  dystrophic  mice,  modified.  It  of  the  more is  it  or  by  a  of  animals in  to  might  would  add  if  both  to  function the  are  major defect i n dystrophic skeletal muscle.  164  a  it  arise  has  2  suggested neurons by  +  PV  probably  though slow  the  PV  observed  in  disturbance  be,  tissue. in  and  that  to  both  electrical PV  the  the  nervous  then A  calcium  and  muscular  the  If  in  in  of  changes  in  trigger  of  the  PV  fire  involved  of  skeletal muscle.  nervous  this  distribution  muscles  conformation  to  in  may  involvement  the  are  PV  neurotransmitters  of  appears in  known,  regulate  the  s p e c i f i c to  expected to  or  Mg  response o f  whether  which  thereby  Even and  of  GABA  of  light  fast  which  credence  enable  and  not  1986)  parvalbumins  study  they  be  In  the  the  release  release  systems.  factor, also  the  tissue.  muscle,  that  and  composition  t i s s u e is  (Celio,  neurons,  determine  P V or  neurogenic  report  GABA  in  interest  -regulated  that  -dependent  unlikely  be  distribution  concentration  2 +  altered  is  would  skeletal  Ca is  global alteration  content PV  PV  dystrophic  regulated  in  2 +  fiber  defect.  nervous  period  proposed  enzymes  neuronal  Ca  recent  Mg-dependent metabolic  P V in  and  protein  is  tissue  represent  a  parvalbumin  changes global  in  its  effect  on  regulation  as  a  BIBLIOGRAPHY A s h l e y , C C . 1983 C a l c i u m i n Wiley, N e w Y o r k , pp. 107-175. 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