Early changes in specific tension following administration of glucocorticoids

MISSING IMAGE

Material Information

Title:
Early changes in specific tension following administration of glucocorticoids
Physical Description:
xi, 60 leaves : ill. ; 29 cm.
Language:
English
Creator:
Eason, Jane M
Publication Date:

Subjects

Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1996.
Bibliography:
Includes bibliographical references (leaves 53-59).
Statement of Responsibility:
by Jane M. Eason.
General Note:
Typescript.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 002117734
notis - AKV7454
oclc - 35730865
System ID:
AA00003183:00001

Full Text









EARLY


CHANGES


IN SPECIFIC TENSION FOLLOWING
OF GLUCOCORTICOIDS


ADMINISTRATION


JANE


EASON


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY


UNIVERSITY


OF FLORIDA


1o o;











DEDICATION


my parents,


Louis


Claire,


teaching


me the


value


an education.












ACKNOWLEDGEMENTS


completion


this


project


would


have


been


possible


without


assistance


of a number


of individuals.


First


would


like


express


my deep


gratitude


mentor


committee


chair


, Dr.


Stephen


Dodd.


am especially


grateful


both


time


guidance


throughout


years


University


of Florida,


as well


as hi


constant


encouragement


throughout


dissertation


phase


my degree


would


also


like


to extend


a spec


thanks


members


doctoral


committee,


Scott


Powers


, Wendell


Stainsby


and


. Daniel


Martin


their


critical


comments


and


advi


to this


manuscript.


would


also


like


to thank


both


Dr. Powers


Martin


allowing


me use


their


laboratories


equipment


as well


as Dr


Darlene


Reid


advice


on the


use


E-64


am particularly


grateful


to the


Foundation


Phys


Therapy


providing


financial


support


previous


proj


ects


leading


to the


dissertation


project.


am thankful


assistance


fellow


student


espe


cially


Sale,


Doug


Robinson


Scott


Stetson


their


assistance


towards


completion


this


project.


r4n~1 Thr I-on A r~rt l-h~,n1rc, nfl, r~ ~ r t\.~ I- c'


C-^ ^ ^ -3 1


r11 n a,


nv q


tk V -dy^T r


I r*"v r






that


anything


possible


with


faith


perseverance.


A big


thank


you


to Neil


enduring


patience


support


throughout


this


project.









TABLE OF CONTENTS



Pace

ACKNOWLEDGEMENTS. . .. ... . . . . I .i i

LIST OF TABLES .. . .... .... . ... . .. . .vil

LIST OF FIGURES .. ...... ...... . . . vSim

S .
ABSTRACT. ..... . . a . . ix

CHAPTER

1 INTRODUCTION.. . a . . . .. . . .1


Statement of the Problem..
Research Hypotheses....
Definition of Terms......
Assumptions .... .. ..a
Liimitations . . ..
Significance of the Study.


. . . . . 6
. a a . a . S . .. 6
.. .a .a. . . .S. . . .7

. . S . . . . 8
. S . . . . . 8
. .. .7.. .
.. . .. . 8


REVIEW OF RELATED LITERATURE........................ 9

Introduction. ... .. .............. .. .. .. ... 9
Morphological Alterations with Glucocorticoid
Treatment on Skeletal Muscle.................. ....10
Glucocorticoids and Body Mass.................. 10
Glucocorticoids and Muscle Mass................11
Glucocorticoids and Muscle Fiber
Cross-Sectional Area .. .. .... ... ... .. 11.
Glucocorticoids and Sarcoplasmic Reticulum.....12
Glucocorticoid-Induced Biochemical Alterations in
SkIeletal) Muscle ... . . . . . . .13
Glucocorticoids and Protein Synthesis......... .13
Glucocorticoids and Protein Degradation........15
Glucocorticoids and Sarcoplasmic Reticulum
Function .. . . .. .. . . 19
Contractile Properties and Glucocorticoids.......20


Cytoskeletal Disruption


as a Possible Mechanism


Leading to a Decrease in Diaphragm Muscle Bundle
Specific Tension . . . . .. .. .. . .22


Use of E-64-c


as a Calpain Inhibitor... ............23









CHAPTER

3 OMATERIAL AND METHODS .... ......a .. .a.. .. ...a 25


Animals .. .. .... . . . .. .. . . . .25
Experimental Des gn. .. . .. . . ..... 25
Experimental Protocol.. ............ 26
Statistical Analysis.......... ...... .. . ... ... 27
Details of Experimental Procedures.................28
Diaphragm Strip in vitro Contractile
Procedures . . . . . . . . .28
Experimental Preparation................... 28
Mechanical Measurements ...................29
Gel Electrophoresis. . . . . . . 29
Whole Muscle Preparation for Gel
Electropho1resis . . .. .. .. .. 29
Titin/myosin Ratio.... .. ..... .. ... a. ... 30


4 RESULTS . . .. .. . . .. .. .. .. .. .. . .32

Morphological Characteristics......................32
In vitro Contractile Properties. ..................33
Titin/myosin Ratio . . . ... .. .. .. . 34


DISCUSSION... .... .. ....... .. ....... ........ 42
Overview and Principle Findings....................42
Body and Muscle Mass Changes with Glucocorticoids..43
In vitro Isometric Twitch Properties...............45
Maximal Tetanic Tension... ... ........ ..............45
Availability of E-64-c to Muscle Cell..........46
Alternative Muscle Protein Degradative
Pathways .. .... ... .. .. .. ... .... .. ... 47
Protein Synthesis/Protein Degradation Rates....50
Summary and Conclusions . .. ... .... ... .. .. .... 51


REFERENCES . a . .. . 53

BIOGRAPHICAL SKETCH .. .. .. .. .. . .. . . 60













LIST


OF TABLES


Table


Morphological
(CTL), predni
(P/E) and E-6


charac
solone
4-c (E)


teristics


PRED)


female


of adult


control


, prednisolone/E-64-c


Sprague-Dawley


rats.


In vitro
control


E-64-c


costal
(CTL),


P/E)


diaphragm s
prednisolone
cd E-64-c (E)


trip


characteristics


(PRED)
female


, prednisolone/
Sprague-Dawley


rats


S ......................S..S*.....- S S S S S


Page















LIST


OF FIGURES


Fiacure


Decline


treatment


body


weight


period...


over
* .


the
. a S f


five


.. .. .... .. ..38


Bar
PRED


graph
. and


depicting
P/E group


titin/myos


>S .


ratio
S*....


for
. .


CTL,
.. .. .. ... 39


Correlational


analysis


relationship


between


costal diaphragm
titin/myosin rat


strip
io....


specific
. .. .. S


tension


- cm


and


Photograph
of costal


of electrophoretic


diaphragm


titin


(SDS- PAGE


myosln


separation


from


E/P,


CTL


PRED


animals . .


Pace


. .. .. ....41












Abstract


Dissertation


Presented


to the


Graduate


School


e University
Requirements


Florida


in Partial


Degree


of Master


Fulfillment


Science


EARLY


CHANGES


IN SPECIFIC


TENSION


FOLLOWING


ADMINISTRATION


OF GLUCOCORTICOIDS

By


Jane


August


. Eason

, 1996


Chairman:


Stephen


Dodd


Major


Department


Exercise


Sport


Sciences


Recent


data


from


our


laboratory


demonstrate


that


administration


glucocorticoids


over


a ten-day


period


results


in a reduction


diaphragmatic


maximal


spec


ific


tension


as well


as a decrease


body


diaphragm


mass


The


cellular


mechanisms


to explain


these


negative


side


ects


remain


obscure.


Elucidating


initiating


mechanisms


of glucocorticoid


-indu


ced atrophy


contractile


dysfunction


be helpful


developing


strategies


prevent


their


occurrence.


purpose


this


study


was


determine


glucocorticoids


would


have


on diaphragm


mass


contractile


function


following


five


days


drug


treatment.


Specifically


, this


study


hypotheses


1) diaphracmatic


maximal


specific


tension


would


be redu


CI


-- v






reduction


in maximal


specific


tension


would


correlate


changes


titin/myosin


ratio.


To test


these


hypotheses,


in vitro


isometric


contractile


properties


were


measured


costal


diaphragm


strips


in adult,


female,


Sprague-Dawley


rats


Animals


were


divided


into


four


groups


: control


(CTL


(n=16),


prednisolone


PRED)


n=13),


prednisolone/E-64-c


P/E)


(n=9


E-64-c


n=6).Animals


were

the


injecte

calpain


with


either


inhibitor


prednisolone


E-64-c


mg/kg


mg/kg


body


body


weight


weight),

every


other


day,


a combination


drugs


or sham


saline


five


days


In addition


, muscles


were


analyzed


titin/myosin


ratio


correlation


to maximal


specific


tension.


results


indicate


that


diaphragm


weight


decreased


11%,


respectively


(p<0


.05)


prednisolone-


treated


prednisolon


-64-c


treated


rats


as compared


control.


Maximal


specific


tension


was


reduced


in the


predni


solone-treat


group


as compared


to control


(p<0


.03)


after


five


days


of treatment.


There


was


no difference


maximal


specific


tension


between


predni


solone


predni


solone


64-c


.No


correlation


(p>0


.05)


existed


between


titin/myosin


ratio.


These


data


support


hypothesis


that


glucocorticoid


treatment


over


five-day


period


results


in a decrease


specific


tension.


However,


this


result


cannot


be explained


changes


in the


titin/myosin


ratio.


probable


then,


-4





atrophy


decreased


force


production


observed


in the


diaphragms


animals


treated


with


glucocorticoids.














CHAPTER 1
INTRODUCTION


Glucocorticoids are widely used


in clinical medicine for


treatment


variety of


diseases,


including


lung


disease


(Wasserman


, 1985)


and connective tissue di


sease


(Askari


et al.,


1976) .


It has been


demonstrated


that


prolonged


treatment


or high doses of


glucocorticoids


result


in muscle atrophy and a decrease


force generation


(Dodd et


al.,


1995


; Vignos,


1976) .


Muscle


atrophy


and a


decrease


force generation may result


oss


functional


capabilities


in patients who are administered


glucocorticoids.


The mechanisms)


to explain


this


dysfunction remain unknown.


Elucidating these mechanisms


will


improve our understanding of how glucocorticoids


alter


both mus


mass


and contractile


properties and will


essential


in developing strategies


to combat


these negative


effects.


Glucocorticoids


affect


cells


in a


manner


characteristic


steroid hormones.


synthesis


In general,


in responsive cells


steroids alter protein


by directly affecting the cell


nucleus.


Specifically,


glucocorticoids


first


enter the







Once


this


binding


occurs,


an activated


hormone-receptor


complex


is created.


This


activated


complex


travels


to the


nucleus


where


binds


directly


spec


ific


DNA


gene


segments


Becker


et al


., 1986;


Groner


et al


, 1984)


This


binding


enhances


transcription


DNA


into


messenger


RNA


units


(Rousseau


1984


Ultimately


messenger


results


increase


d prot


ein


synth


s in


cell.


newly


formed


proteins


can


alter


cell


function


acting


as enzymes


membrane


carriers


structural


proteins.


In contrast


excessive


eve


of glucocorticoids


seen


in clinical


treatment


inflammatory


diseases


Cushing


s di


sease


or in experimental


animal


, result


mus


tissue


molecular


catabolism


mechanisms


atrophy


to explain


Unfortunately


hormone-indu


ced muscle


wasting


remain


unclear.


It has


been


demonstrated


that


animal


s injected


with


glucocorticoids


over


an 8-


to 14-day


period


experience


a 14-


decline


body


mass


(Sasson


et al


., 1991;


Dodd


et al


1995;


Lieu


et al


1993


Moore


et al., 1989;


Viires


et al


1990


Further


asson


et al.


1991


erved


that


60-70%


the animal


s' weight


loss occurred


in the


first


week


We have


made


similar


observations


our


laboratory


as well


Rats


injected


with


a dose


mg-kg


-1. day


prednisolone


experience


rapid


weight


oss


first


days


treatment.


In the


days


following


this


rapid


weight


oss


, as







loss of


weight,


so that


tenth


injections


, body


weight


begins


to plateau


Lieu


et al.,


1993) .


decrease


body


weight


is due


to a decre


ase


prot


ein


synthes


, an increase


in protein


degradation


or a


combination


two.


time


course


response


prot


ein


synthesis


degradation


over


a 12 day


period


corticosterone


admini


station


skeletal


muscle


was


examined


Odedra


et al


1983


results


indicated


that


overall


the

was


first


rate


four

the


prot


days so

initial


ein synthesis


that


rate.


This


in muscle


five,

lower


decreased


protein

d rate


over


synthesis

was


maintained


following


week.


These


changes


in the


rate


protein


synth


esis


were


accompanied


a transient


increase


in prot


ein


degradation.


The rate


protein


degradation


more


than


double


ed by


two


three


treatment,


returned


to the original


rate


This


suggests


that


rapid


body


weight


oss


seen


in the


first


days


glucocorticoid


administration


to a simultaneous


decrease


in prot


ein


synthe


a transient


increase


prot


ein


degradation.


five


six,


daily


body


weight


oss


is not


as great


, and


s is


likely


to the


continued


depre


ssion


rate


protein


synthesis


, while


protein


degradation


returned


to its


original


rate.


mechanisms


to explain


transient


increase


protein


degradation


remain


unclear


However


, Kayali


and







administration


in rats.


Calpain


is a calcium-dependent


cyst


eine


protease


that


ubiquitous


in a


variety


tissues


and


cell


Melloni


& Pontremoli,


1989)


Calpain


located


near


the Z-di


muscle


cell


is apparently


responsible


degrading


Z-di


Busc


et al


1972


Other


studies


have


reveal


ed that


calpain


very


limited


effect


on contractile


proteins


Goll


et al., 1992)


Therefore


, it


seems


that


calpain


can


play


a role


muscle


protein


degradation


exc


lusively


directing


effects


toward


cyto


skeleton,


rather


than


myofibrillar


protein,


which


results


in disassembly


myofibril


ease


large


polypeptide


fragments.


cytos


calpain


keletal


s called


protein


titin.


Titin


specifically


a large


affected

protein


x 106


Maruyama


et al., 1984)


comprises


approximat


total


myofibril


mass


(Wang


et al.,


1984;


Trinick


1984


Evidence


from


several


laboratory


indicate


that


titin


molecule


links


thick


filaments


to Z-di


scs


skeletal


musci


(Maruyama


et al


., 1985;


Trini


et al


1984


Gassner,


1986).


Consequently


, the


initial


effects


of glucocortoicoid


administration


on protein


degradation


result


from


increase


in calpain


activity


which


degrades


titin


ultimately


disas


sembles


intact


myofibrils


into


filament


polypeptide


filaments.


There


are


several


lines


of evidence







glucocorticoid


treatment.


First,


60-70% of


total


body


weight


loss occurs


in the


first


four to six days of


glucocorticoid treatment.


Second,


an increase in calpain


activity has been noted with glucocorticoid injections


(Kayali


& Young,


1985) .


Finally,


it has been demonstrated


that


calpains are specific


the breakdown of


cytoskeletal


protein


titin)


and not myofibrillar protein.


Since


titin


connects


the thick filament


to the Z-disc,


the degradation of


titin would result


the disassembly of


intact myofilaments


from Z-discs.


The selective


loss of


titin from skeletal muscle cells


also be implicated in


glucocorticoid-treated animals.


force decrement


Evidence


observed in


from several


laboratories have shown


conclusively that


titin links


thick


filaments


1984


to Z-di


; Gassner,


scs


1986) .


in skeletal muscles


Horowitz and Podol


Maruyama


(1988


al.,


showed


that


the physiological


function of


titin


filament


prevent


sarcomere asymmetry


from occurring over repeated


isometric


between


contractions


contractions as well


by recentering the


as to keep


thick


thick


filament


filaments


centered during passive stretch


Further,


when


titin


filament


selectively removed from single skinned fibers


with low doses of


muscle


ionizing radiation,


to generate both active and passive


the ability of


tension is greatly


reduced


(Horowitz et al.,


1986) .


Potentially then,







tension


in whole


muscle.


seems


then,


that


important


to determine


a decrease


in tension


generation


occurs


early


stages


glucocorticoid


administration


and


what


role


potential


loss


of titin


play


Statement


of the


Problem


Patients


use


glucocorticoids


treatment


pulmonary


disease


, connective


sue


sease


or who


undergo


ransplantation


organ t

atrophy


in protein


may


contractile


degradation


be at risk


dysfunction.


observed


developing

transient


in experimental


muscle

increase


animals


indicates


that


this


dysfunction


an early


development


in a


treatment


time


regime


course


glucocorticoids.


mechanisms


of contractile


Identification


dysfunction


initial


step


developing


strategies


towards


ameliorating


problem.


Research


Hvootheses


maximal


tetanic


isometric


specific


tension


in costal


diaphragm


muscle


bundles


will


be decreased


glucocorticoid


treated


rats


over


a five


course


of drug


treatment


compared


to control


diaphragm


muscle


bundles.


decrease


isometric


tension


seen


in costal


diaphragm


muscle


bundles


of glucocorticoid


-treated


rats


to activation


of calpains


will


correlate


with


changes







Definition of Terms


Diaphragm muscle bundles are strips of


diaphragm muscle which


are attached to a


force


transducer and stimulated via a pair


platinum electrodes while


immersed in an


in vitro organ


bath.


Titin refers


to a


large cytoskeletal


protein


that


comprises


about


10% of myofibril mass.


Functionally,


important


re-centering the myosin molecule between Z-discs


following a


contraction.


Fluorinated alucocorticoids are a class of


synthetic


glucocorticoids with 9-alpha


fluorination.


They


are


generally


believed to be more myopathic than nonfluorinated


glucocorticoids.


Dexamethasone is


prototypical


this class


of glucocorticoids.


Nonfluorinated alucocorticoids


are a


class of


synthetic


glucocorticoids which does not have


9-alpha


fluorination as


part


its chemical


structure.


Prednisolone


is prototypical


of nonfluorinated glucocorticoids used to


treat


human


disease.


Soecific


PO represents


the absolute


tetanic


tension generated


the diaphragm muscle


strips divided by the cross-sectional


area


the strip.


This allows


comparison between muscle


strips of


differing


sizes.







Assumptions


sample


study


are


female


representative


Sprague-Dawley


of the


rats


population


used

all


in this

female


Sprague-Dawley


rats.


Glucocorticoids


are


similarly


metabolized


rats.


There


are


no differences


between


Krebs-Henseleit


homogenization


or gel


electrophoresis


solutions


study


progresses.


Limitations


invasive


nature


this


study


precludes


use


human


subjects.


Sianificance


Study


Diaphragm


muscle


atrophy


loss


strength


administration


of glucocorticoids


significant.


focus


previous


inve


stigations


has been


on long-terms


ects


.e 8


-30 days


of glucocorticoids.


unknown


muscle


atrophy


and


function


occur


earlier.


In addition,


these


unwanted


side


effects


are


to be prevented,


appears


necessary


to investigate


mechanisms


which


may


be the


initiating


factors


as well


as the


time


course.


Therefore,


result


these


this


initiating


study


factors


useful


ultimately


towards


designing


elucidating


therapeutic














CHAPTER


REVIEW


OF RELATED


LITERATURE


Introduction


is generally


well


accepted


that


administration


high


doses


of glucocorticoids


a short


period


time


result


in muscle


atrophy


a de


crease


in fo


rce


production


diaphragm mus


Dodd


et al., 1995;


Moore


et al.,


1989;


Sasson


et al


primary


., 1991) .

inspiratory


Because


music


diaphragm

decrease


considered


in force


generation


may


have


eterious


effects


in patients


with


lung


sease.


Understanding


underlying


mechanisms


contribute


to these


side


effect


benefit


cial


designing


strategies


to prevent


their


occurrence.


This


review will


focus


on the


changes


in skeletal


muscle


function


with


gluc


ocorticoid


administration


with


particular


attention


to the


time


course


these


changes


Specifically,


will


focus


on adaptation


diaphragm muscle


since


use


of glucocorticoids


prevalent


patients


who


have


respiratory


sease.


Finally,


potential


mechanisms


which


may


explain


force


deficit


seen


in muscles


from


glucocorticoid-treated


animals


will


be examined.







Moroholocical


Alterations


with


Glucocorticoid


Treatment


Skeletal


Muscle


Glucocorticoids


Body


Mass


Most


studies


report


a decrease


body


weight


animals


following


gluco


cortic


treatment.


Admini


station


cortisone


acetate


mg-kg


*day


10 day


resulted


decrement


in body


weight


as compared


to a control


group


of Sprague-Dawley


rats


Moore


et al.,


1989) .


Likewi


Dodd et

treated


al. (

with


1995


predni


reported


solone


a 22%


body


mg-kg


weight


*day


loss


a ten


animal

day


riod.


Furthermore


other


investigators


report


a 22-40%


body


weight


oss


with


varying


dosages


time


increments


glucocorticoid


admini


station


Viires


et al


1990;


Sasson


1991


Dekhui j zen


et al.,


1995;


Lieu


et al


time


courses


changes


body


weight


are


very


rapid.


Sasson


colleagues


1991


observed


that


60-70%


total


weight


OSS


during


week


glucocorticoid


treatment


period


occurred


in the


first


week.


Further


Peter


et al.


1970


report


an initial


weight


oss


10-12


first


24 hours


after


one


injection


triamcinolone


continuous


weight


10 days.


loss


our


g/day


laboratory,


over


we have


treatment


seen


similar


period


result


unpubli


shed


consistent


observations


with


changes


These


in protein


observations


synthes


are


s/degradation


*rr l anI ,-' -' -* 1 t r f4 -


rf-m: f nI VI rc C /- I' 1 ^t-t-


^ -^\ t- l >/-


n-,^ v.I r


u7 ^*-/- ;n


nC -^ -


/-h t


*-"







than


reported


literature.


These


ideas


will


expanded


upcoming


protein


synthesi


s/degradation


section.


Glucocorticoids


Muscle


Mass


Consistent


glucocorticoid


with


treated


decrease


animals


body


, parallel


weight


decrements


diaphragm


muscle


weight


have


been


reported


several


investigators


Administration


of prednisolone


10 days


a dosage


of 5


mg-kg


*day


resulted


a 15%


decre


ase


diaphragm


music


weight


(Dodd


et al


., 1995) .


Collectively,


several


mass


investigators


18-42%


have


animal


report


s treated


ed decreases


with


in diaphragm


glucocorticoids


varying


doses


and


time


increments


(Dekhuijzen


et al.,


1995;


Lieu


et al.


, 1993


Moore


et al.,


1989;


Viires


et al., 1990).


To dat


one


, all


Dekhuij zen


these


et al.,


inves


1995),


tigations


have


, with


focused


exception


on treatment


regimens


no less


than


eight


days


with


most


being


in the


range.


Given


rapid


body


weight


oss


observed


initially


with


glucocorticoid


admini


station,


seems


logical


that


this


should


hold


true


diaphragm


muscle


mass


as well.


Glucocorticoids


Muscle


Fiber


Cross-Sectional


Area


Gluco


corticoids


preferentially


affect


less


active







spared


the atrophic effects of


glucocorticoids.


The effects


of glucocorticoids on diaphragm muscle


fiber cross-sectional


area have been described in several


animal


studies and


results are contrary to


the notion


that


the diaphragm would


be spared from the effects of


glucocorticoids.


Significant


atrophy of


type


fibers


was


shown


the coastal


diaphragm of hamsters


treated with dexamethasone


.5 mg. kg.


over


days


(Lewis


et al., 1992).


Further,


a 44%


54% decrease


in both


type


fibers


as well


as a


decrease in


type IIa


fibers was


reported in


the diaphragm


of rabbits


treated with cortisone acetate


(10 mg-kg-1-day-1)


weeks


(Ferguson et al.,


1990)


Finally,


a 51% decrease


in type


fibers with no change in cross-sectional


area


type


IIa or type


fibers


following administration of


triamcinolone


(0.5 mg-kg-day-1)


been reported by


Dekhuijzen


et al


(1995)


Overall,


previous


investigations


overwhelmingly support


the observation


that


administration of


glucocorticoids


results


in significant


atrophy


type


fibers.


The degree of


atrophy appears


to be dependent


animal


species,


route


of drug administration,


type of


glucocorticoid used


(fluorinated


vs.


non-fluorinated)


, as


well


length of


time


the drug


is administered.


Glucocorticoids and Sarcoolasmic Reticulum


Few studies have examined the effects of


glucocorticoids


1 .day-1







ten-day


period


resulted


disruption


sarcoplasmic


reticulum


in the


extensor


digitorum


longus


(EDL).


disruption


loss


SR occurred


areas


where


Z-line


streaming


was


noted.


Further


, the


was


frequently


more


prominent


glucocorticoid-treat


than


in controls


with


localized


dilation


SR tubules


in some


fibers


However,

membrane


atrophy

systems


EDL


muscle


remain


fibers


same


also

size,


occurred


SR will


appear


dilated.


In contrast


Peter


et al. (


1970


suggest


that


sarcotubular


ves


icles


isolated


from


rats


injected


with


-1 day


triamcinolone


days


not


show


abnormalities.


This


conclu


sion


was


reached


biochemical


SR rather


assays


than


measuring


electron


calcium


microscopy.


handling


However,


ability


was


concluded


that


if calcium


handling


was


not


affected,


then


structural


abnormalities


of the


SR would


an expected


result.


Clearly,


there


a paucity


literature


examining


structure

function


and

will


glucocorticoids.


be discussed


Biochemical


biochemistry


changes


in SR


section


this


review.







Glucocorticoid


- Induced


Biochemical


Alterations


Skeletal


Muscle


Glucocorticoids


Protein


Svnthesi


Ther


is general


glucocorticoids


decrea


agreement

se protein


in the 1

synthes


literature


It has


hat

been


shown


that


decrease


in prot


synthesis


can


be due


impairment


peptide


chain


initiation


Rannel


& Jefferson,


1980;


Rannel


et al., 1978)


Additionally,


Goodlad


and


Clark


1991


demonstrated


termination


an increase


transcripts


which


in the


would


frequency


impair


of premature


elongation


stage


transcription


ultimately


decrease


rate


transcription.


glucocorticoids


on both


protein


the

1986


ribosomal


content


result


was examined

this study


revealed


EDL (

that


Kelly

part


et al


atrophic


effect


glucocorticoids


arises


from


an inhibition


protein


synth


esis


decrea


ses


in the


ribosomal


capacity


music


which


ultimately


to a decrease


protein


content


muscle


Odedra


et al. (


1983


demonstrated


that


glucocorticoids


to a fall


to a loss of RNA.


overall


protein


rate


synthesi


of protein


primarily


synthesis


was


initial


rate


fourth


corticosterone


treatment


This


depressed


rate


of synthesis


tt., C' r4 4- .^ 4^ n>^ A 41A ,


10


=rm4 ni


c? t- rz- in


Ea otrQr


i y 1 -


n^F / -~ "


T- ^n i







atrophic


effects


glucocorticoids


may


attributed


depres


sed rates


protein


synthes i


in the


early


stages


drug


administration


which


maintained


throughout


course


treatment.


Glucocorticoids


Prote


in Degradation


overall


consensus


on the


role


glucocorticoids


protein


degradation


remains


unc


lear


Previous


investigations


have


shown


that


prot


ein


degradation


s increase


Tomas


1979


Pennington,


decreased


1977)


McGrath


, unchanged


& Goldspink,


Ranne


1982


& Jefferson


Shoj i


1980


undergoes


a biphasi


response


Odedra


et al


1983


as a


result


glucocorti


coid


administration.


Whil


differing


dosages


, routes


admini


station


time


length


glucocorticoid


section


cannot


counted


, the


main


source


different


these


discrepancies


techniques


measure


s lik


musci


to the


proteolysis


use


as well


a short


timecourse


protein


degradation.


method


measuring


myofibrillar


protein


degradation


(Engel


is to


& Franzini


measure


-Armstrong,


urinary


levels


1994


of N-methylhistidine


an amino


acid


found


primarily


actin


also


s located


myos


of white


music


zsar


, 1972


It


derived


from


posttranslational


modification


stidine


residues


when


is generated


proteoly


, it cannot


re-incorporated







contractile


proteins.


examining


total


protein


proteolysis


release


labeled


tyrosine)


as well


as myofibrillar


proteoly


N-methylhistidine


production)


, investigators


can


determine


glucocorticoid


effects


on both


non-myofibrillar


and


myofibrillar


protein.


Several


investigators


have


demon


state


an increase


urinary


3-methylhistidine


pharmacological


doses


as result


of glucocorticoids.


administration


increase


methylhistidine


production


peak


ed by


of drug


treatment


and


gradually,


over


following


to 5


returned


seline


level


Toma s


et al.,


1979;


Santidrian


et al


1981)


using


indirect


methods


measuring


musci


proteolysi

transient


Odedra


rise


et al. (


in protein


1983)


demonstrated


degradation


glucocorticoid


treatment


followed


a return


to ba


seline


eve


over


next


several


days


Kayali


et al.


1987


examined


both


urinary


mus


-methylhistidine


level


well


tyros ine


ease


in i


isolated


muscle


found


similar


results


Glu


cocorticoid


treatment


res


ulted


in an increase


3-methylhistidine


excretion


urine


which


was


followed


treatment.


a return


This


to ba


parallel'


seline

d the


level


release


3-methylhistidine


from


perfused


hindquarter


in treated


rats


However


, rel


ease


tyros ne


from


perfu


sed hindquarter


treated


rats


was


ected


gluc


ocorticoid


treatment.


Based


upon


basal


F -I 1 1


S1 _


CI rl


-I


r







breakdown


of myofibrillar


protein


that


breakdown


nonmyofibrillar


protein


is regulated


differently.


However


these


studi


are


examining


final


product


in the


pathway


protein


breakdown


overlook


role


that


protease


activity


play


in early


stages


of protein


degradation.


increase


demonstrated in


in alkaline


gastrocnemius


protease


muscle


rats


activity


treated


was


with


glucocorticoids


Mayer


et al


, 1974


; Mayer


et al., 1980)


well


from


quadriceps


Seene


et al.,


1988) .


However


Clark


Vignos


1981)


not


report


an increase


alkaline


prot


ease


activity


psoas


mus


of rabbit


treated


with


glucocorticoids


two-week


period


reflect


a species


difference


between


rats


rabbits.


SOS


omal


proteases


was


examined


in a study


Clark


and


Vignos


1981) .


Injection


of betamethasone


mg-kg


-1 day


weeks


result


ed in


decrease


psoas


type


fibers


music


weight


New


Zealand


white


rabbits.


Concomitant


with


decrease


in muscle


weight


was


a significant


increase


in catheps


D activity.


No de


crease


in soleus


weight


was


found


, and


likewi


, in that


music


there


then,

protE


was


that


no increase


lysosomal


degradation


in cathepsin


proteases


with


D activity.


play a

admini


glucocorticoid


seems


role


station


There


activat


been


ed protea


ses


much


interest


play


in the


in protein


role


cium-


breakdown.


Calpain







& Pontremoli,


1989).


It has


been


demonstrated


that


calpains


have


a very


specific


on myofibrillar


proteins


Goll


al., 1992).


Incubation


myofibrils


with


purified


calpain


presence


calcium


resulted


complete


removal


Z-line

at the


Goll


et al.,


peripheral


19


part


92) -

of the


Since


myofibril


s the

s that


myosin

are i


filaments


initially


eaved


in an atrophy


process


Seene


et al.,


1988


, it


seems


logical


tulate


that


destruction


Z-line


calpain


atrophy.


would


Indee


an initiating


, an increase


process


in the


leading


pool


music


of released


filament


with


was


calcium


demonstrated


ionophore


when


A23187


solat


and/or


ed muscle


calpains


treated


van


Westhuyzen


et al


, 1981).


Several


investigators


have


examined


activity


calpains


glucocorticoid


-treated


muscle


which


have


yielded


conflicting


res


ults


Clark


Vignos


1981


found


increase


in calpain


activity


psoas


muscle


rabbits


ated


with


Young


glucocorticoids


1985


demonstrated


weeks.


an increase


However,


calpain


Kayali


activity


in rat


gastrocnemius


fourth


of corticosterone


treatment,


while


there


was


no increase


catheps


activity


during


same


time


riod.


calpains


are


initiator


of myofibrillar


breakdown


, it


seems


reasonable


assume


that


calpain


activity


would


increa


early


treatment


period


rather


than


at a later


period


This


may







increase


calpa in


activity


shown


Kayali


and


Young


1985


with


administration


glucocorticoids


as well


as the


with


increase


cium


in relea


ionophore


filaments


A23187/calpain


from muscle


seems


treated


to support


notion t

register


hat


a cytoskeletal


undergoes


limited


protein

proteoly


that

sis.


keeps

This


filaments


proteolysis


leads


an increase


in released


filaments


which


ultimately


results


muscle


atrophy.


Glucocorticoids


Sarcoolasmic


Reticulum


Function


role


factors


of sarcoplasmic


contributing


reticulum


to glucocortico


dysfunction

id-induced


as one

muscle


weakness


was


examined


a study


Shoj i


et al.


(1976)


Calcium-dependent


ATPase


activity,


calcium


uptake


binding


sarcoplasmic


reticulum


were


significantly


reduced


vastus


lateralis


muscle


isolated


from


rabbits


treated


with


triamcinolone


acetate.


While


functional


implications


impaired


biochemical


properties


sarcoplasmic


reticulum


were


not


examined,


could


speculated


that


a decrease


force


generation


muscle


would


a consequence.


Additionally,


impairment


cium


handling


be the


signal


that


activates


cium-activated


neutral


prot


eases


which


then


leads


muscle


atrophy.


However,


Peter


et al.


1970


found


no difference


I


I







rats


treated


with


triamcinolone


acetate


. Clearly,


there


a consensus


regarding


effect


glucocorticoids


have


sarcoplasmic


reticulum


function


.The


conflicting


conclusions


between


species


these


and


studies


duration


could


treatment


a result


differences


of dosage,


An additional


diff


erence


could


muscle


sampled.


Shoj i


et al.,


1976


used


vastus


lateralis


which


has a high


proportion


type


fibers


, while


Peter


et al


1970


used


more


evenly


mixed


gastrocnemius.


Contractile


Prooerti


Glucocorticoids


The

properties:


literature


examining


diaphragm


from


the c

animal


changess


treaty


in contractile

ed with


glucocorticoids


reports


mixed


result


Studies


have


shown


change


in specific


tetanic


tension


(Lewis


et al.,


1992


Dekhuij


zen


et al.,


1995) ,


a decrease


in abso


lute


tension


, but


cifi


tension


Moore


et al


1989


, as well


as a


decr


ease


in both


measurements


Sasson


et al.,


1991


, Dodd


1995


The


literature


reports


mixed


res


regarding


twitch


parameters


as well.


Regarding


specific


twitch


tension,


several


glucorticoid


investigator


treated


animals


report


as compared


no change


to control


Dekhuijzen


et al.


, 1995


Lewi


et al


Viires


et al.,


1990


while


others


report


a decr


ease


in specific


twit


tens:


glucocorticoid


treated


animals


Moore


et al







animals


1992;


(Sasson


et al.,


Viires et


al.,


1991;


Moo


1990) .


re et al., 1989;

A prolongation c


Lewis et


)f one-half


relaxation


time has


been reported by several


investigators


(Dekhuijzen et al.,


1995


Sasson et al.,


1991)


while others


have observed no change


Lewis et al.,


1992;


Moore et


al.,


1989


Viires et


al.,


1990)


Further


, an increase


in the


twitch:tetanic tension ratio has been reported


(Sasson


al.,


1991


.The


increase in both the twitch:tetanic


tension


ratio and one-half


relaxation


time appears


to indicate


that


changes


in the calcium-handling ability of


the sarcoplasmic


reticulum may have occurred as a result


of glucocorticoid


administration.


considering


Comparison of


studies


type and dosage of


administration as well


difficult


glucocorticoid used,


as the length of


time


route


the drug was


injected are different.


However,


the overall


consensus


appears


indicate


that


glucocorticoids


can affect


diaphragm


contractil


examined


function.


the effects of


Furthermore,


glucocorticoids


these studies


from a period


eight

Odedra


days


et al.


weeks


(1983)


of glucocorticoid administration.


showed that


protein


synthesis/degradation rates


return


to baseline levels


by the


12th day


of glucocorticoid treatment.


This


indicates


that


muscle protein loss seen initially


in glucocorticoid


administration has


returned to a


level


whereby myofibrillar


protein


loss


is no


longer occurring.


Since Odedra


et al.


al.,







glucocortiocoid


administration,


seems


logical


examine


contractile


measurements


in the


initial


days


glucocorticoid


injections.


ctqrntni n


5s a Pnss


Mechanism


Leadina


Decrease


in Dianhracrm Muscle


Bundle


Soecific


Tension


selective


loss


titin


from


skeletal


muscle


cells


may


be implicated


in the


force


decrement


observed


glucocorticoid-


treated


animals.


Titin


a large


protein


whi


ch links


thick


filaments


to Z-di


scS


skeletal


muscle


(Maruyama


demonstrate


et al.


that


, 1985) .


titin


Titin


reaches


antibodies


from


have


M-line


been


to the


used


Z-line


(Trinick,


titin


1994).


A band


Furthermore,


does


appears


appear


that


to stretch,


portion


while


portion


located


in the


band


appears


to be elastic


(Fulton


Isaacs,


1991) .


Higuchi,


Destruction


or by


titin


ses


either


ioni


zing


digestion


radiation


(Horowits

isometric


et al.,

tension


1986)


results


in skinned


single


a decrease

fibers.


in active

Furthermore,


loss


titin


does not


appear


to affect


actin-myosin-


regulatory


proteins


interaction


(Higuchi,


1992) .


Electron


microscopy


generation


shows


that


primarily


decrease


appears


to be


in active


to di


tension


sordering


structures


within


sarcomere


(Higuchi,


Loss


titin


may


also


play


a role


disassembly


^-. ,- 4 A,-.


r n n


snVrfltn 4 Tt


~ I ~ ,e IUL*Iryi U I ru~evw~, v lrr -


Cvtoskeletal


__ ^ -


-^- h -k~ Tn n t^- C^-


rUAr.


Mlnh







series


proteolytic


event s


occur,


myofibrils


would


have


to be


disas


sembled.


Since


titin


attaches


thick


myosin


to the


Z-line,


loss of


titin


would


result


in rel


ease


myofilaments


which


could


then


be degraded


role


as a cytoskeletal


filament


, it


seems


then,


that


proteolysis


of titin


calpain


may


initial


step


in the


oss


as well


turnover


myofibrillar


myofibrillar protein

as a decrease in the


proteins


would

force


result


Ultimat


in muscle


generating


ely,


t rophy

of the


ability


muscle


Use


of E-64


as a Caloain


Inhibitor


A wide


variety


of agents


have


been


used


in an attempt


inhibit


actions


calpain.


However


, many


these


agents


also


inhibited


actions


many


other


proteases


were


membrane


permeabi


Recently,


use


of E-64


demonstrate


some


success


in inhibiting


calpains


E-64

inhibitors


a member


of calpain


of a cl


ass


isolated


microbial

from As


peptide


pergi ili


j aponl cus


(Parkes


et al


1989


Besides


inhibiting


calpain,


E-64 al


inactivates


cathepsins


B and


Barrett


1982


Although


membrane


permeance


E-64


poor,


synthetic


analogues


have


proven


to be


membrane


permeabi


Shoji-Kasai


et al., 1988)


ability


of E-64


to inhibit


calpain


activity


been







was


then


incubated


with


E-64.


Calpain


activity


was


decreased


after


a 40 minute


incubation


period.


Sugita


et al


1980


injected


E-64


mg-kg


subcutaneously


twice


a week


months


in control


and


dystrophic


chicks.


While


calpain


activity


in E-64-treated


strophi c


chicks


was


greater


than


normal


control,


activity


was


that


untreated


dystrophi c


chicks.


Spontaneous


locomotor


activity


normal


dystrophic


mice


was


measure


over


week


period


conjunction


with


receiving


E-64


(Komatsu


et al.,


1985) .


Three-week-old


control


trophic


mice


rece


ived


either


E-64


mg-kg


1.day


E-64


mg-kg


-day


, P.o.


or no inhibitor


weeks.


decrease


in locomotor


activity


normally


seen


untreated


dystrophic


mice


was


retarded


E-64


E-64-d


treated


dystrophic


mice.


Additionally


, survival


time


was


prolonged


in treated


dystrophic


mice


Tracheal


banded


hamsters


injected


with


E-64


-c at


dosage


calpain


mg/kg


activity


every

v afte


other

r only


day

two


resulted


treatments


a 70%

with


decrease

E-64-c (D


Reid,


personal


communication).


Furthermore


, soleus


mus


incubated


simultaneously


with


both


calcium


ionophore


A23187


and


E-64-c


showed


a 14%


reduction


in protein


degradation


compared


to medium


without


E-64-c


Zeman


et al.


, 1985


Based


increase


on the


in calpain


above


evidence,


activity


was


seems


that


responsible


initial







increase.


Furthermore,


inhibition


of titin


degradation


with


E-64-c


should


correlete


with


changes


in titin/myosin


ratio


between


groups


as seen


in SDS-polyacrylamide


electrophoresis.
















CHAPTER


MATERIALS


AND


METHODS


Experimental


Design


Animals


Adult


4-month-old


female


Sprague-Dawley


rats


were


used


these


experiments


.The


invasive


nature


ese


experiment


precluded


use


of human


subjects.


Utili


zing


this


strain


rats


allows


comparison


to other


studies


since


as well


as others


have


used


Sprague


-Dawley


rats


previous investigations


Experimental Desian


Forty-four


examine


female


diaphragm muscle


electrophoresi


s for


Sprague-Dawley


strip


titin/myos


rats


contractile


ratio.


were


used


properties


animal


and


were


individually


house


chow


water


libitum


while


being


maintained


on a 12-hour


light/dark


photoperiod


prior


to beginning


expe


riments.


During


-day


period,


animals


were


handl


ed daily


to reduce


contact


stress


animal


were


assigned


one


four


experimental


groups.








Group


Treatment;


daily


prednisolone


injections


mg/kg


days


(n=13


PRED)


Group


Treatment;


daily


prednisolone


sections


mg/kg


E-64


injections


mg/kg


every


other


days


Group


Control


E-64


injections


mg/kg


every


other


days


(n=6


Predni


solone


was


suspended


saline


injected


subcutaneously


daily


days.


Predni


solone


was


chosen


because


is prototypical


of glucocorticoids


used


treat


human


sease


E-64-c


was


sso


ved in


a phosphate


buffer


and


injected


singly


subcutaneou


in conjunction


every


with


other


predni


solone


days


either


sections


sections


were


done


at approximat


same


time


.The


control


animals


receiving


predni


solone


were


sham


injected


with


saline


days


project


was


approved


University


of Florida


Institutional


Review


Board.


Guidelines


animal


use


establi


shed


American


Phys


biological


Society


were


followed.


Experimental


Protocol


Animals


were


euthani


zed by


intrape


ritoneal


injection


sodium


pentobarbital


mg/kg


the entire


diaphragm


removed


placed


in a di


ssecting


dish.


A small








end.


strip


were


used


to determine


in vitro


contractile


measurements


time-to


; spec


ifically,


-tension


peak

TPT)


isometric

one-half


twitch


tension


relaxation


time


, and


contractile


Following


peak


isometric


measurements


were


measurements


tetanic


collected


, the


mass


tension


with


music


Po)


music


strip


well


as the


length


was


determined


calculation


muscle


cross


-sectional


area


CSA)


utilizing


formula:


CSA


=muscle


mass


/[muscle


length


x mus


dens


g/cm3


Assuming


muscle


density


.056


g/cm3


Metzger


& Fitts


, 1986


remaining


costal


diaphragm


was


trimmed


of connective


tissue


fat,


blotted


weighed


on an analytical


balance


was


then


frozen


in liquid


nitrogen


stored


-70


measurement


of titin


myos


heavy


chain


profile


Stati


stical


Analvsi


Compari


sons


between


each


expe


rimental


group


day


prednisolone


injections


of only


E-64-c or


simultaneous


E-64-c


predni


solone


sections


control


group


were


made


one-


factor


ANOVA


followed


appropriate


significant


post-hoc


different


testing


ces


occurred.


order


to determine


following


where


pairs


comparisons


were


chosen


as contrasts


a pri ori:


Group


vs.


Group


and


Group


vs.


Group


The relations


hip


between








Significance


contrast


was


tests


established


, significance


at p<0.05


was


established


ANOVA.

d at p<0


For

.03.


Details


of Experimental


Procedures


Diaohraam


String


vitro


Contractile


Measurements


Experimental


Dreparation.-


After


reaching


a surgical


plane


anesthesia


, the


diaphragm


was


removed


and


placed


a di


ssecting


sh containing


a Krebs-Henseleit


solution


equilibrated


with


a 95%


.The


muscle


strip


was


ssecte


d from


ventral


costal


region


suspended


vertically


between


two


plexiglas


clamps


connected to


force


trans


ducer


Cambridge


Technology


, model


300B)


in a


jacketed


tissue


bath


containing


Kreb


s-Hense


leit


solution


SIn


order


to produce


complete


neuromuscular


blockade


, 12 M


tubocurarine


was


added


to the


tissue


bath.


keted


tissue


bath


was


aerate


d with


5%C02


, pH


maintain


at 7


.4 and


osmolality


bath


was


-290


mOsmol


Temperature


in the


organ


bath


was


maintained


at 250C.


The muscle


strip


was


stimulated


along


entire


length


with


platinum


wire


ectrodes


using


a modified


Grass


Instruments


stimulator.


A supramaximal


voltage


was


use


volts


to stimulate


strip


After


15 minute


equilibration


riod


music


strip


was


adjusted to


maximal


twitch


tension


obtained.


This


was


acc


omplished


applying


I1 4 -


- .


*


1 (





*







Mechanical


Measurements.


Cambridge


transducer


output


was


amplified


differentiated


operational


amplifiers


undergo


conversion


analysis


using


computer


Peak


based data


isometric


acquisition


twitch


system


, time-to-peak


truments


tension


eries


TPT)


half


relaxation


time


(RT1/


was


determined


computer


analysis


force


transduced


output.


Additionally,


peak


isometric


tension


was


produce


ed by


using


a supramaximal


stimulus


train


of 80 Hz and


msec


duration.


Electroohoresis


Whole


muscle


oreoaration


electroohoresi


Myofibrillar


protein


was


isolated


a modification


myofibril


extraction


procedure


as described


Solaro


et al


1971


Approximately


costal


diaphragm


designat


ed for


myofibrillar


prot


ein


analy


S1S


was


thawed


cleaned


tendon


minced


with


a scissors


minced


muscle


was


homogeni


zed in


ml of


sucrose


buffer


centrifuged


min.,


at 2500


rpm.


supernatant


was


discarded


pellet


resuspended


a KC1


buffer


containing


Triton


-X 100


pellet


was


centrifuged


again


15 min


.at


rpm;


this


step


was


then


be repeated.


supernatant


from


third


spin


was


scared


pellet


resuspended


a KC1


buffer


centrifuged


again;


s step


was


then


repeated.


final


pellet


was


II) .







concentration.


The biuret


protein concentration


technique was used to measure


(Watters,


1978).


Titin/Mvosin Ratio.


The myofibrillar protein was


diluted in sample buffer


.5 mM Tris


pH=6.8),


1.0%


SDS,


0.01% bromophenol blue


, 15.0%


glycerol


, 5.0% 6-


mercaptoenthanol,


of -20 mg/ml.


-950 C


The prot


for three minut


.04 rM leupeptin


ein was


es.


to a


then denatured b


The entire volume wa


concentration

y incubation at

s loaded onto


cm vertical


s (Biorad


Protean IIxi)


composed of


a 4%-


10%-15%


layer resolving


system as described by


Frit


(1989)


This


type of


loading system allows


eas


observation of


changes


in electrophoretic migration with very


high-molecular weight


proteins


(Fritz


et al,


1989)


The


samples were electrophoresed


24 hours at


-50 C using a


constant


voltage


required to attain an


initial


current


milliamps per gel.


Following


electrophoresis,


gels were


stained with Coomassie Blue R-250


The relative proportion of


myosin heavy


chains,


as well


as the


titin bands were


analyzed using a


computer-based image analysis


system


Targa


image capture board,


Truevision;


Mocha


video analysis


software,


Jandel


Scientific)


integrated with a high


resolution CCT video camera


Video World


, Inc.) .


The amount


titin was normalized relative


that


of myosin since


is difficult


to accurately control


the amount


protein


loaded onto


the gels


(Granzier


& Irving,


1995).













CHAPTER
RESULTS


Moroholooical


Characteristics


Physical


characteristics


of the


animal


are


summarized


in table


Mean


body


weights


between


groups


beginning


study


were


different


(p>0


However


following


five-day


injection


period,


mean


body


weights


animal


s treated


only


with


prednis


olone


PRED)


were


lower


.05)


than


body


weights


control


CTL)


animals


lower


than


E-64-c


animal


Likewise


, the


body


weight


animal


s treated


with


both


predni


solone


E-64-c


P/E)


were


decrea


sed by


.05)


as compared


to the


(CTL


group


lower


.05)


than


the E


group


There


were


no differences


between


PRED


.03).


Figure


illustrates


decrease


body


weight


over


five-day


treatment


period


Closer


examination


reveal


that


group


showed


a significant


decrease


body


weight


second


treatment


while


predni


solone


treated


d animals


experience


a significant


decrease


(p<0O


body


weight


until


third


treatment


Body


weight


PRED


P/E groups


on the







There


were


no differences


in the


body


weights


between


PRED


groups


throughout


treatment


.03)


diaphragm


weights


paralleled


body


weight


changes


predni


over


solone


five


-treated


-day


group


treatment


experience


period


a 15%


table


decrease


.05)


while


group


an 11%


decrease


diaphragm


weight


as compared


to CTL.


Compared


to the


group


diaphragm


weights


PRED


group


were


lower


.05)


while


group


experienced


decrease


.05)


in diaphragm


weight


There


was


no diff


erence


diaphragm


weight


between


PRED


groups


(p<0


.03)


No changes


in musci


weights


of either


soleus


or the


extensor


digitorum


longus


.05)


were


noted


in any


group


In vitro


Contractile


Properties


Maximal


spec


tetani


tension


was


lower


.03)


predni


solone


group


as compared


to control


Tabl


No different


ces


.03)


maximal


spec


ific


tetanic


tension


existed


between


the predni


solone


predni


solone


groups.


Table


illustrates


vitro


twitch


contractile


characteristics


the diaphragm music


strips


There


were


diff


erences


cific


twitch


tension


between


PRED


was


greater


P/E


(p>0


.05)


as compared


However


to CTL


(p<0


.05)


in the


group


was


, Pt








Time


to peak


tension


development


(TPT;


time


required


reach


maximum


isometric


force)


was


prolonged


group


as compared


to the


PRED


group


.03)


as well


group


There


were


no differences


between


CTL


PRED


groups


with


regards


TPT


.03)


nor


was


different


from


other


three


groups


(p>0


.05).


No differences


.03)


existed


between


vs.


PRED


PRED


vs.


groups


with


respect


to RTI/2


(RT1/2;


time


necessary


force


to decrease


from maximum


to one-half


maximum)


However,


group


was


greater


than


PRED


group


(p<0


.05)


was


greater


than


CTL


group


(p<0


.05)


was


different


from


group


.05)


regards


to RT1/2.


ratio


was


different


(p>0


.03)


in the


CTL


PRED


or PRED


. P/E


groups.


However,


Pt/Po


was


greater


(p<0


.05)


in the


group


as compared


to the


group


greater


was


greater


than


.05)


PRED


group


group


Similarly,


as compared


to CTL.


Finally


, no differences


(p>0


existe


d in


between


Titin/mvosin


ratio


No differences


.05)


existed


between


CTL,


PRED


or P/E


groups


regards


to titin/myosin


ratio


data


from


group


unavailable


Figure


Values


are


follows


(mean+SEM)


RT1/2


Pt/Po


Pt / Po


Pt/Po







between Po and


titin/myosin ratio was


0.012


(r=0.11)


significant


correlation


(p>O


.05)


existed between Po and


titin/myosin ratio


(Figure 3)










Table 1


Morphological


characteristics of


adult


control


(CTL),
(P/E)


prednisolone
and E-64-c (E


PRED),


prednisolone/E-64-c


female Sprague-Dawley rats


Parameter CTL PRED P/E E


Initial body weight


Final body weight


280.3


280.8


285.6


9c,b


287.1
+5.4


Costal
weight


diaphragm


(mg)


598.7
+11. 5


5a,b


c,b


EDL weight


(mg)


121.6


115.4


+9.6


+1.5


Soleus weight


(mg)


115.5
+2.8


120.0


128.0


129.0
+7.4


Values are means+SEM


sample


EDL,


extensor digitorum longus


Variables with contrasts
a p<0.03, significantly


different


from CTL


Variables with post-hoc tests
b p<0.05, significantly different


from E


p<0.05


, significantly


different


from CTL


la,b










Table


In vitro costal


control


E-64
rats


(CTL),
P/E) a


diaphragm strip characteristics


prednisolone
mnd E-64-c (E)


(PRED)
female


, prednisolone/
Sprague-Dawley


Parameter CTL PRED P/E E


n 13 11 9 6

Po (N-cm-2) 21.4 18.6 18.6 19.9
+.76 +.728 +1.1 +1.4

Pt (N-cm-2) 5.7 5.6 6.1 7.8
+.2 +.5b +.6 +.5d

RT1/2 (msec) 50.0 52.7 59.4 64.0
+2.5 +2.0b +2.8 +_2.0d

TPT (msec) 53.0 52.7 62.4 55.8
+1.8 +2.0c +2.9d +2.9

Pt/Po 0.27 0.30 0.35 0.40
_+.01 +.02b +_.02d +.03d


Values are means+SEM


sample


size;


maxinal


specific


isometri


tetanic


tension
tension
tension


Newtons;


; RT1I


one-half


peak


isometric


relaxation


time


twitch specific


TPT,


time


to peak


Variab:
a p<0


1


with


contrasts


.03, significantly different
.03, significantly different


from CTL
from P/E


Variables with post-hoc tests
b p<0.05, significantly different
d p<0.05, significantly different


from E
from CTL


; N,


































-r
-
- -K.
-
- a
- -- -I----- - .~-.---,iI--
-.- -
-
-1-


EIo- -l

Control


- _---


Pred


Pred/E-64-c


N


0-
a


I, I I I I


. I I I


I 1 I I


I


Figure


Decline
period.


in body weight


over


five


treatment


significantly
significantly


different
different


from
from


L (p<0.
(p<0.05


1 I I I















































PRED


Figure


PRED


graph
, and


depicting
P/E groups


titin/myosin


ratio


CTL,






















































SI I I


a I I


S I l I


i.l I


I l I


I 1. I


Ia I


I t I I


Titin/myosin


Ratio


Figure


Correlational


costal


diaphra


titin/myosin


analysis
gm strip


of the
specific


relationship
c tension (N


between


*cm-2)


ratio


* CTL

A PRED

0 P/E


w




















Titin--->


Myosin--->


1


2 ::



2


3













CHAPTER


DISCUSSION


Overview


PrinciDle


Findings


This


study


tested


hypotheses


specific


glucocorticoid-induced


atrophy


contractile


dysfunction


observed


a decrease


cos


in the


diaphragm.


specific


first


tension


hypothesis


of diaphragm


was


muscle


that


strips


would


occur


after


five


days


of glucocorticoid


administration.


Secondly,


was


hypothesized


that


decrease


in specific


tension


would


be correlated


to changes


titin/myosin


ratio


could


be prevented


with


a calpain


blocker,


E-64-c.


Results


indicate


that


a decrease


in specific


tension


occurs


in diaphragm muscle


strips


after


five


days


glucocorticoid


admini


station.


However,


results


show


that


crease


in spe


cific


tension


is not


related


to changes


titin/myosin


ratio


or is


block


ed by


E-64-c.


Thus,


appears


that


activation


calpains


may


not


play


significant


role


atrophy


decrement


force


generation


observed


animals


treated


with


glucocorticoids.







Body and Muscle Mass Chanaes with Glucocorticoids


This


study


is unique in


that


first


to document


changes


diaphragm muscle mass and body weight


after short-


term


(five days)


, high doses


of glucocorticoids


experimental


animals.


Most


studies


literature have


reported changes


these variables


after


10 or more days


(Lieu et al.,


1993


Dodd et


al.,


1995;


Sasson


al.,


1991;


Ruff


et al.,


1982)


results observed in


this


study may be clinically


relevant


because


the dose administered and


time course


of administration.


asthmatics


Acute myopathy has


been reported in


hospitalized with severe exacerbation


their


disease and requiring high doses


intravenous


glucocorticoids


short


periods of


time


(Van Marle &


Woods,


1980


Knox


et al.,


1986;


Williams


et al.,


1988).


Furthermore,

short-term,


ventilators


difficulty i

high doses of


n weaning patients


treated with


intravenous glucocorticoids


been reported


(Van Marle & Woods,


from


1980


Williams et al.,


1988;


Knox et


al .,


1986) .


Although


diaphragm mass


in these patients


could not


be measured for


obvious


reasons,


possible that


a decrease


in muscle


mass


resulted in a


OSS


of absolute


force generating ability


the diaphragm.


inability of


This may have been a


these patients


potential


to be weaned


factor


from the


ventilator.


A loss


in diaphragm


strength has been shown







glucocorticoids


Weiner


et al.,


1993;


Decramer


et al.,


1994


There


was


no difference


between


groups


in regards


soleus


weight


This


was


an expected


finding


since


glucocorticoids


have


been


shown


to have


a preferential


effect


on fast


fiber


types


Clark


& Vignos,


1979;


Ruff


et al.


1982)


interesting


finding


was


that


mass


extensor


digitorum


longus


EDL)


muscle


was


affected


glucocorticoids


this


study


In contrast


, Ruff


et al.


1982


three


reported


days


a significant


of dexamethasone


decrease


injections


in EDL


while


mass

corti


after

sone


acetate


inje


actions


result


in significant


mus


atrophy


until


after


seven


days


treatment.


possible


explanation


could


be due


to differences


between


types


glucocorticoids


used


both


studies


Dexamethasone


fluorinated


glucocorticoid


as opposed


to both


cortisone


acetate


prednisolone,


which


are


non-fluorinated


glucocorticoids -


Fluorinated


glucocorticoids


generally


are


more


myopathi


than


non-fluorinated


glucocorticoids.


appears


that


from


this


study


least,


that


diaphragm


was


more


sus


ceptible


to the


atrophic


effects


short-term,


high


doses


of prednisolone


as compared


to the


EDL.


mechanism (


A loss


to explain


in muscle


this


are


body


not


weight


clear 4

could


this


time.

to changes


mus


1993


water


showed


ontent

that


or protein


reduction


content.

muscle


However


mass


Lieu


with


m~ -










can


explain


results


this


study


Lieu


1993


show


decr


eases


in the


protein


content


of muscle


from


glucocorticoid


-treated


animals


which


would


indicate


enhanced


protein


degradation.


vitro


Isometric


Twitch


Prooerti


Overall


examination


result


s indicate


that


section


of E-64-c


alone


con junc


tion


with


predni


solone


resulted


prolongation


several


twitch


characteristic


In general,


section


either


E-64


-c alone


or in


conjunction


with


predni


solone


resulted


in prolongation


most


twitch


contractile


chara


cteris


Tabi


these


contractil


character


stics


are


indicative


calcium


handling


muscle


refl


changes


both


calcium


uptake


as well


as rel


ease.


No studi


exist


the literature


examining


direct


effect


of E-64-


on contractile


properties


of muscle


Determination


factors


res


ulting


prolongation


many


twitch


characteristics


observed


are


beyond


scope


this


study,


appears


that


combination


of predni


solone


E-64-c


or E-64


alone


exert


changes


on calcium-handling


abiliti


muscle


Maximal


tetani


tension


report


s the


first


to document


a decrease


in in







This


finding


seems


consistent


with


clinical


reports


patients


experiencing


difficulty


resuming


spontaneous


respiration


despite


resolution


of broncho


spasm


following


short-term


treatment


with


glucocorticoids


(Van


Marle


& Woods,


1980


Knox


et al.


, 1986


Williams


et al.,


1988


Another


hypothesis


this


study


was


that


decrease


specific


tension


would


be a result


degradation


titin


to activation


of calpains.


to its


role


as a


cytosk


eletal


filament,


proteoly


of titin


calpain


may


result


dismantling


contractile


proteins


from


Z-disc


may


be the


initial


step


turnover


of myofibrillar


proteins.


Indeed


, loss


titin


been


shown


cause


decrease


spec


ific


tension


single


fibers


(Higuchi,


1992;


Horowits


et al.,


blocking


1986) .

actions


We proposed


calpains


to test


this


proteas


hypothesis

e inhibitor


E-64-c


examining


treatment


the titin/myosin


glucocorticoid


-inj ected


ratio.


animal


However,


with


co-


E-64-c


prevent


ecrease


in Po


(Table


nor


it prevent


decrease


in muscle


weight


(Table


There


are


several


possibilities


that


explain


lack


of effect


E-64-c


to prevent


glucocorticoid-induced


atrophy


decrease


force


production.


Availability


of E-64-c


to muscle


cell


first


possibility


that


inhibitor







to CTL


Table


indicate


that


E-64-c


crossed


sarcolemma


sufficient


quantities


to antagoni


actions


calpains.


Furthermore,


our


proto


was


same


as one


used


in another


study


E-64-c-treated


. Reid,


tracheal


personal


-banded


communication)


hamsters


showed


in which


decrease


calpain


activity


diaphragm


as compared


to those


who


received


no inhibitor.


ated


animal


s received


subcutaneous


injections


E-64-c


mg/kg


body


weight


every


other


redu


action


in calpain


activity


was


observed


third


treatment


protocol.


Alternative


muscle


protein


dearadative


oathwavs


Second,


several


biochemical


pathways


intra


cellular


sites


prot


ein


degradation


poss


ible


that


one


or more


these


pathways


are


response


ible


decrease


in muscle


mass


as well


decr


ease


seen


animals


treated


with


glucocorticoids.


main


sites


muscle


prot


eoly


are


cytoplasm


lysosomes


Bird


et al


., 1980)


Furthermore


cytosolic


pathway


include


calcium


-dependent


proteases


calpains


, an ATP


-dependent


system


the proteasome


multi


-catalytic


protease).


Briefly,


-dependent


pathway


involves


a prot


cofactor


multienzyme


ubiquitin


process


which


activated


Ubiquitin


an ATP-requiring


s conjugated


to proteins







ubiquitin


from


protein


that


subsequently


degraded


Tawa


suggest


& Goldberg


that


, 1994) .


calcium


Furthermore


only


regulates


, evidence


exists


calpain


which


system,


proteasome


catalytic


system


as well


Realini


Rechsteiner,


1995).


Recent


evidence


indicates


that


glucocorticoids


may


necessary


to stimulate


ATP-dependent


system.


Wing


Goldberg


1995


were


unable


to demonstrate


significant


role


calpain


proteolyt ic


system


fasting


rat s


nor


were


they


able


to demonstrate


change


in the


ability


muscles


conditions


to activate


which


both


process.


Indeed


lysosomal


, under


calcium-activated


systems


were


blocked,


dexamethasone


was


administered


fasted,


adrenalectomized


rats


Results


indicated


that


rate


of muscle


proteolysis


was


increased


level


fasted,


normal


animals


only


after


addition


glucocorti


coids


(Wing


Goldberg,


1995)


Furthermore


, mRNA


eve


Is of poly-Ub


were


increased


opposed


fold


to fasted,


in music


fasted,


adrenalectomized


animals


control


(Wing


animal


& Goldberg,


1995).


Tiao


et al.


1996


also


demonstrated


an increase


free


ubiquitin


as well


levels


muscle


ubiquitin


mRNA


rats


treated


with


dexamethasone


Tiao


et al,


1996) .


Blocking


glucocorticoid


receptors


with


RU38486


inhibited


ubiquitin


and


ubiquitin


mRNA


levels


(Tiao


et al,


1996) .


This


provides







likely


that


protein


degradation


under


conditions


increase


ed lev


of glucocorticoids


involves


coordination


seven


proteolytic


pathway


Indeed


incre


ased


mRNA


levels


catheps in


, calpain


ubiquitin


been


report


ed in


rats


treated


with


dexamethasone


Darcevet


decreases


would


et al


1995


in music


be due


.Our

body


to activation


hypothesis


weight


was


as well


calpains


that

as de


Base


early

crease


on the


above


evidence


several


appears


proteolyt ic


likely


that


pathways.


glucocorticoids


poss


then,


activates


that


calpain


system


plays


only


a small


role


muscle


degradation


observed


with


coc


orticoids


this


s true


it would


expe


cted


that


blocking


calcium-activated


system


with


E-64-c would


not


an effective


countermeasure


to the


glucocorti


coid


-induced


atrophy


observed


this


study.


Another


possibility


to explain


inability


of E-64-c


to blunt


effects


of glucocorticoids


in this


study


could


that


calcium


enhanced


proteoly


SiS


occurs


increase


synthes i


PGE2


rather


than


activating


calpains


Rodeman


and


Goldberg


1982


demonstrated


that


muscles


incubated


medium


containing


cium


ionophores


resulted


enhanced


rates


of protein


degradation


; however,


when


calcium-activated


prot


eases


were


blocked


there


was


decrease


in prot


ein


degradation


Furthermore


, they


demonstrate


ed that


produce


tion


was


enhan


ced 3


fold







blocking


sosomal


proteases


with


inhibitors


added


calcium-enriched


medium


inhibited


enhanced


protein


breakdown


normally


seen


Rodeman


& Goldberg


From


series


experiments


, they


concluded


that


incre


ased


intracellular


calcium


enhanced


protein


degradation


through


effect


on lysosomal


function


which


was


mediated


prostaglandins


However


, several


inves


tigator


dispute


notion


that


elevated


degradati

Turinsky


level


of PGE2


Barnett


& Loegering


res


& Elli


, 1985)


increa


1987;


In all


Clark

three


rates


et al.,


thes


protein


1984;

e studies


proteoly


S1S


was


not


acce


lerated


with


increase


d leave


PGE2


seems


then


that


a clear


does


not


exist


between


PGE2


increased


rates


of proteolys


Despite


inconsistency


in studi


involving


PGE2


prot


ein degradation,


poss


ible


that


elevated


level


PGE2


may


have


play


some


mus


atrophy


and


decreased


specific


tension


observed


this


study


Protein


svnthesi


s/orotein


degradation


rates


third


possibility


that


depression


protein


synthesis


play


s a


more


important


role


than


protein


degradation


does


condition.


literature


contradictory


regarding


relati


contribution


of protein


synthesis


degradation


rates


to the


music


wasting








synthesis


explains


body


muscle


weight


loss


seen


animal


s treated


d with


glucocorticoids


Ranne


Jeffer


son,


1980;


Rannels


& Jeffer


son


, 1978;


McGrath


& Gold


spink,


1982


Shoji


& Pennington,


implicates


protein


1977)


However


degradation


as the


evidence


major


exists


component


which

in the


music


wasting


seen


in conditions


exhibiting


excessive


glucocorticoid


level


Kelly


& Goldspink


, 1982;


Kayali


al., 1987;


Konagaya


et al.,


1986;


Odedra


et al


1983


Despite


evidence


regarding


glucocorticoid


-induced


prot


eolys


, it


appears


that


precise


mechanisms


explain


catabolism


remain


unclear.


Summary


Conclusions


These


glucocorticoids


experiments


would


sought


have


to determine


on diaphragm muscle


effect


mass


contractile


function


after


only


five


of drug


treatment.


data


demon


state


that


diaphragm mass


force


production


are


decreased


following


only


five


days


glucocorti


coid


injections.


Further


, it


was


hypothes


ized


that


decrease


in mass


isometric


tetanic


tension


would


degradation

degradation


titin

titin


molecule.

molecule


The

does


res

not


ults


indicate


explain


that


loss


mass


was


rce-


evidenced


generating


no change


ability


diaphragm.


titin/myosin


ratio


This


between


groups


fact


that


no correlation


existed


between







play


a role


in the


early


atrophy


decreased


force


production


observed


in the


diaphragms


of animals


treated


with


glucocorticoids.









REFERENCES


Askari,


Vignos,


and R.


myopathy in connective disease
492, 1976.


Moskowitz.
Am. J.


Ie


Steroid
d. 61:485-


Barrett,
M.


Kembhavi,


Tamai,


and K.


Hanada


leucylamido(4-guanidino


inhibitors of


cathepsins


Brown,


Kirschke,


. L-trans-Epoxysuccinyl
)butane (E-64) and its


cysteine proteinases


H and L.


Biochem.


Knight,


analogues


including
210:189-198,


1982.


Barnett,


Ellis.


Prostaglandin E2


regulation


of protein degradation in skeletal muscle.


Muscle Nerve.


10:556-559,


1987.


Becker,


Gloss,


Schmid,


In vivo protein-DNA interactions


response element


Nature.


Strahle, and G. SchOtz.
in a glucocorticoid


require the presence of


324:686-688,


the hormone.


1986.


Busch, W. A., M
specific
muscle. J


. H.


Stromer,


removal
. Cell B


of Z lines


iol


Goll,


and A.


from rabbit


52:367-381,


Suzuki.
skeletal


Ca2+


1972.


Clark


and P.


experimental


Vignos.


role of proteases


glucocorticoid myopathy.


Muscle Nerve.


4:219-222,


1981.


Clark


, A. S., R
to thermal


Kelly,


and W.


injury to rats.


Mitch.


. Clin.


Systemic response


Invest.


74:888-897,


1984.


Dardevet, D.
Attaix,


response
muscle


Sornet,


and J.


Grizard.


Taillandier,
Sensitivity


I. Savary,
and protein


to glucocorticoids are different


from adult


and old rats


. J.


Clin.


turnover


in skeletal
Invest.


96:2113-2119,


1995.


Decramer,


Lacquet,


Fagard,


and P.


Rogiers.


Corticosteroids contribute to muscle weakness


airflow obstruction.


. Resoir.


Crit.


in chronic


Care Med.


150:11-16,


1994.


Dekhuijzen
Bock,


N.
Dom,


SG.
and M.


Gayan-Ramirez,
Decramer. Co


A. Bisschop, V. De
:ticosteroid treatment


~j2 f-c -----J-. L ~ -


-^ a .. A-


*I -


1 -- _. -r








Dodd,


Powers,


S. Vrabas,


and J.


Eason.


Interaction of
affect muscle


glucocorticoids and activity patterns
function. Muscle Nerve. 18:190-195, 1995.


Ferguson,


. Irvin,


and R.


Cherniack.


Effect


corticosteroids on respiratory muscle histopathology


Rev.


ResDir.


Dis.


142:1047-1052,


1990.


Fritz,


Swa rt z,


and M.


. Greaser.


Factors


affecting polyacrylamide gel
electroblotting of high-molec


proteins.


Anal.


Biochem.


18


electrophoresis and
ular- weight myofibrillar
0:205-210, 1989.


Fulton,


and W.


Isaacs.


Titin,


a huge,


elastic


sarcomeric protein with a probable role


morphogenesis.


BioEssavs.


13:157-161,


in
1991.


Gassner,


Myofibrillar


purified antibodies


interaction of blot


against


native


immunoaffinity-


titin as


studied by


direct


immunofluorescence and


immunogold staining.


Eur.


Cell.


Biol.


40:176-184,


1986.


Goldberg,


and H.


Goodman.


Relationship between


cortisone and muscle work in determining muscle size.


Phvsiol.


(London)


a 200:667-675,


1969.


Goll,


Thompson


Christiansen. Role o
growth. Biochimie.


, R.


Taylor,


and J.


f the calpain system in muscle
74:225-237, 1992.


Goodlad,


and C.


Clark.


muscle atrophy:alterations
of skeletal muscle nuclei.


Glucocorticoid-mediated
transcriptional activity


Biochim.


Bioohvs.


Acta.


1097:166-170


1991.


Granzier,


and T.


muscle: Contrib
and intermediate


Irving.


ution of c
filaments


Passive tension


ollagen, titin,
- BioDhvs. J.


in cardiac


microtubules,
68:1027-1044,


1995.


Groner,


Kennedy,


DNA sequences
expression by


involved


Skroch,


Hynes,


regulation of


glucocorticoid hormones.


and H


Ponta.


gene


Biochim.


BioChvs,


Higuchi,


781:1-

Change


digestion of


froa


6,


1984.


s incontractile properties with selective
connection (titin) in skinned single fibers


skeletal muscle.


Biochem.


111:291-295,


1992


Acta.







Horowits,


and R.


Podolsky


Thick


filament movement and


isometric tension in activated skeletal muscle.


Bioohvs.


54:165-171,


1988.


Horowits, R.,
Podolsky.


. S. Kempner, M
A physiological


skeletal muscle.


Nature.


. E.


Bisher,


role for


and R.


titin and nebulin


:160-164,


1986.


Huszar,


Developmental


changes ofthe primary


structure and


histidine methylation in rabbit


skeletal muscle myosin.


Nature


(New Biolo.y)


240:260-264,


1972.


Jaspers,


and M.


in the response


Muscle Nerve.


9: 554-561,


Tischler


. Rol'


leg muscle


e of glucocorticoids
to reduced activity.


1986.


Kayali, A
Ca2+


and V.


Young


. Effect


corticosterone on


- activated protease and cathepsin D activity in


gastrocnemius.


Fed.


Proc.


44:1895,


1985.


Kayali,


Young,


myofibrillar proteins
proteolysis. Am. J.


and M.


Goodman.


Sensitivity of


to glucocorticoid-induced muscle


Phvsiol.


252:(Endocrinol.


Metab.


15):E621-E626,


1987.


Kelly


and D.


four muscle
Biochem. J.


tvoes


. Goldspink. The
to dexamethasone


208:147-151,


differing responses of


treatment


in the


rat.


1982.


Kelly, F.
Culle


J. J. A. McGrath, D. F. Golds
n. A morphological/biochemical


of corticosteroids on rat


pink,


study


skeletal muscle.


and M.


on the actions
Muscle Nerve.


9:1-10,


1986.


Knox,


Mascie-Taylor,


hydrocortisone myopathy
41:411-412, 1986.


and M.


Muers.


A


in acute severe asthma.


.cute
Thorax.


Komatsu,


effect


K. Inazuki,
of new thiol


derivatives,


Hosoya,


protease


on dystrophic mi


Satoh.


inhibitors,
ce. Exp. NE


Beneficial


epoxide


_urol.


91:23-29,


1986.


Konagaya, M., P. A. Bernard, and S. R. Max. Blockade of
glucocorticoid receptor binding and inhibition of


dexametha
RU38486,


sone-induced muscle atrophy


a potent


EndocrinoloCav


in the rat


glucocorticoid antagonist.


119:375-380,


1986.








Lieu,


Wood,


Powers, R.
Stainsby,


Herb,


and C.


Criswell,


Chen.


Martin,


glucocorticoid-induced diaphragmatic myopathy.


Phvsiol.


75:763-771,


1993.


Maruyama, K.,
Kikuchi.


Kimura,


Yoshidomi,


Molecular size and shape of


elastic protein of


striated muscle.


. Sawada,


and M.


S-connectin,
. Biochem.


95:1423-1433,


1984.


Maruyama, K
and R.


and Z


Natori.


lines


Connection


Higuchi,
filaments


. Ohashi,
link thick


* Kimura,
filaments


immunoelectron microscopy.


Cell Biol.


101:2167-2172,


1985.


Mayer


, M., R. Amin, and E. S
of proteolytic activity


hafrir


skeleta


in response
1 and


diaphragm muscles
Mol. Cell. Endocr


to hormones


inol.


1980.


Mayer, M., R.
protease:


Amin,


and E.


Shafrir.


Rat myofibrillar


Arch.


Biochem.


161:20-25,


1974.


McGrath,


and D.


Goldspink.


Glucocorticoid action on


protein synthesis
skeletal muscles


and protein breakdown in


Biochem.


206:641-645,


isolated


1982.


Melloni,


Pontremoli.


The calpains.


TINS.


12:438-


444,


1989.


Metzger,


and R


Fitts.


Contractile and biochemical


properties of diaphragm: e
fatigue. J. ADDpl. Physiol.


effects of


exercise


1986.


Moore,


Miller,


Feldman,


and M


. B.


Reid.


Diaphragm atrophy and weakness
rats. J. ADol. Phvsiol. 67:24


2


in cortisone-treated
0-2426, 1989.


Murachi, T.,
calpain


Intracellular regulatory system involving
and calpastatin. Biochem. Int. 18:263-294,


1989.


B. R., P.
the effect


protein
K _


turnover


Bates,


in rat


and D.


Time course


corticosterone on


Exercise and


Yoshioka,


in frog skeletal muscle as


revealed by


Differences


in cardiac,


18:49-58,


and catabolic conditions.


conditions of muscle protein degradation.


enzyme properties and adaptive changes


60:1752-1758,


Odedra,


catabolic doses of


Millward.


skeletal muscle and liver.


AoPl.


I


_ I __







Parkes,


Kembhavi,


by peptide epoxides.


and A.


Barrett.


Biochem.


Calpain


230:509-516,


inhibition


1985


Peter


A. Verhaag,


steroid myopathy.


and M.


Examination of


Worsfold.


Studies of


the possible effect


triamcinolone on mitochondria and sarcotubular vesicles


of rat


skeletal muscle.


Biochem.


Pharmacol.


19:1627-


1636,


1970.


Rannels,


and L.


Jefferson.


Effects of


glucocorticoids on muscle protein turnover


in perfused


rat hemicorpus.


Phvsiol.


(Endocrinol.


Metab.


1_:E564-E572


1980.


Rannels, S. R.,
Jefferson.
initiation


Rannels,


Pegg,


and L.


Glucocorticoid effects on peptide-chain


in skeletal muscle and heart.


Phvsiol.


235:E134-E139


, 1978.


Realini, C.
subunit


and M.


Rechsteiner


binds calcium.


A proteasine activator


Biol.


Chem.


270:29664-29667,


1995.


Rodeman,


Waxman,


and A.


Goldberg.


stimulation of


protein degradation in muscle by Ca2+


mediated by prostaglandin E2 and does


calcium-activated protease.


Biol.


Chem.


require the
257:8716-


8723,


1982.


Rousseau,


hormones.


. Control
Biochem.


gene expression by glucocorticoid


224:1-12,


1984


Ruff,


Martin,


induced atrophy
rat muscle. Am.


and A.


is not


Phvysiol.


Gordon.


Glucocorticoid-


to impaired excitability of


(Endocrinol.


Metab.


6L:E512-E521,


1982


Santidrian,
Effect


Moreyra,


Munro,


of corticosterone and its


and V.


route of


R. Young.
administration


on muscle


protein breakdown,


measured in vivo by urinary


excretion of N-methylhistidine in rats:response


different
Metabolism


levels of


30:798-


dietary protein and energy.
804, 1981.


Sasson,


Tarasiuk,


Heimer


, and H.


Bark.


Effect


dexamethasone ondiaphragmatic and soleus muscle
morphology and fatigability. Respir. Phvsiol.


85:15-


1991.








Shoji,


and


on protein
muscle. Mo


Pennington.


breakdown


. Cell


synthesis


. Endorinol


effect
in rat


:159-169


cortisone


skeletal


, 1977


Shoji,


Takagi,


of sarcoplasmic


myopathy.


H. Sugita,
reticulum i


. Neurol


Toyokura.


rabbit


51: 304-309


human


Dysfunction
steroid


, 1976.


Shoji-Kasai, Y., M.
Thiol protease


epidermoid


Senshu,
-specifi


carcinoma


C


A431


. Iwashita
inhibitor


Imahori.


E-64


at mitotic


arrests


metaphase.


human
Proc.


Natl


. Acad.


. USA.


85:146-150


, 1988


Solaro, R. J.,
of cardiac


. C. Pang
myofibril


and I
with


Triton


riggs.
X-100


The purification
. Biochim.


Bioohvs


. Acta


245:259-262,


1971


Sugita,
of


. Ishiura


epoxide


neutral
Biochem.


derivatives


protease


(CANP)


87:339-341,


Suzuki,


on chicken
In Vitro


Imahori.


Inhibition


calcium-activated


In Vivo.


1980.


Tawa


Goldberg


Protein


and


amino


acid


metabolism


muscle.


McGraw-Hill


Mvolocrv:


Philadelphia.


Basic
1994.


Clinical


Tiao


, G., J.
Fischer


Fagan


. Roegner,
Hasselgren.


Lieberman,


. Wang,


. E.


Energy-ubituitin-dependent


muscle proteolysis
glucocorticoids.


during
. Clin.


sepsis
Invest


in rats


97:339-348


regulated


1996


Tomas, F. M., H
glucocorti
protein br


. N.
coid


Munro


administration


eakdown


V1VO


Young.


on the


in rats


Effect


rate


as means


of muscle


ured


urinary


excretion


of N- Methylhi


stidine


Biochem.


:139-146


1979.


Trinick,


Titin


nebulin:


protein


rulers


in muscle?


TIBS


19:40


5-409


, 1994.


Turinsky,


.and


. Loegering.


Prostaglandin


muscle


protein
Biochim


turnover
. BioDhvs


in Pseudomonas


Acta.


840:137-140,


sepsis.


1985


Van


Marle, W.,
myopathy.


L. Woods


Med.


Acute


281:271-272,


hydrocortisone


1980


aerug nosa


Ed.)







van der Westhuyzen,


K. Matsumoto,


Easily releasable myofilaments
muscles maintained in vitro.


and J.


from skeletal


Biol.


Chem.


. Etlinger.
and cardiac
256:11791-


11797,


1981.


Vignos,


and R.


skeletal muscle


Lab.


Clin.


Greene.


Oxidative respiration of


in experimental


Med.


81:365-378,


corticosteroid myopathy.
1973.


Vignos, P. J., A.
properties of


. Kirby,
rabbit


and P.


fast and


H. Marsalis.
slow muscles


Contractile
in steroid


myopathy.


Exo.


Neurol


53:444-453,


1976.


Viires,


Pavlovic,


. Pariente,


of steroids on diaphragmatic


and M.


function


Aubier.


in rats.


Effects
Am. Rev.


ResDir.


Dis.


142:34-38,


1990.


Wang,


Ramirez-Mitchell,


extraordinarily


protein.


Proc.


long,
Natl.


and D


flexible,


Acad.


Palter.


Titin i


and slender myofibrillar


_ 81:3685-3689,


1984.


Wasserman,


Conn's Current


Therapy.


Saunders.


Philadelphia.


1985.


Watters,


A one-step biuret


assay


for protein


in the


presence of


detergent.


Anal.


Biochem.


88:695-698,


1978.


Weiner,


Azgad,


and M.


Weiner


. The effect


corticosteroid on inspiratory muscle


performance


humans.


Chest.


104:1788-91,


1993.


Williams,


O'Hehir


, D.


Czarny,


Home,


and G.


Bowes.


Acute myopathy in severe acute asthma


treated


with intravenously administered corticosteroids.


Am.


Rev.


ResDir


. Dis.


137:460-463,


1988.


Zeman,


Etlinger
calcium.


Kameyama,


Regulation of


Biol.


Chem.


Matsumoto,


protein


Bernstein,


degradation


260:13619-13624,


and J.


in muscle


1985











BIOGRAPHICAL


SKETCH


Jane


Eason


was


born


raised


Orleans,


Louisiana.


graduated


from


Loui


slana


State


University


Medical


Center


in May


1979


with


a bachelor's


degree


physical


therapy.


rehabilitation


Jane


from


Loui


received


siana


a master


State


s degree


University


in cardiac


Baton


Rouge,


LA in


1983.


In 1991,


she began


graduate


study


toward


degree


Doctor


Philosophy


exercise


phys


iology


with


a minor


physiology


University


Florida.


Following


graduation,


Jane


will


a post-doctoral


associate


Anatomy


Cell


Biology


Department


at Emory


University,


Atlanta,


GA under


direction


Dr. Arthur


English.






I
opinion


certify


that


if conforms


presentation a
a dissertation


have


read


to acceptable


fully


adequate,


degree


this


study


standards


in scope


Doct


and-that in my
of scholarly


and quality
ilosoptf7'


Step
Asso


ate


. Dodd, C
Professo


hair
r of


Exercise


and Sport Sciences


certify


my opinion


that


it conforms


presentation a
a dissertation


is fully


have


read


to acceptable


adequate,


degree


this


study


standards


in scope


of Doc


that


of scholarly


and quality
ilosophy.


, as


Scott


K. Powers


Prof


essor


Exercise


Sport


Sciences


certify


that


have


read


this


study


that


in my


opinion


it conforms


presentation a
a dissertation


to acceptable


fully


adequate,


degree


standards


in scope
tor of P


of Doc


of scholarly
and quality,
hilosoh.,. ,^


A. Daniel Martin


Associate
Physical


Professor
Therapy


certify


opinion


that


conforms


presentation a
a dissertation


have


read


to acceptable


fully


adequate,


degree


,is study
standards


in scope


of Doctor


that


of scholarly
and quality,


Philosophy


Wendell N. Stains


Professor


of Physiology


, as







This


dissertation


College


Graduate


School


requirements


August


was


of Health


and
for


was
the


submitted


Human


accepted
degree


1996


to the


Performan


as partial
f Doctor o


(X^


nnd


Col:


Graduate
ce and t


Faculty
o the


fulfillment
f Philosophy.


leae of Healt


h


Human Performance


Dean,


Graduate


School
















LD
1780
199k,







UNIVERSITY OF FLORIDA
II1 1 1 1 IIIII IIIII
3 1262 08553 8824