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Physiological, molecular and biochemical characterization of rodent extraocular muscles: Implications for Duchenne Muscular Dystrophy [Elektronische Ressource] / Ulrike Zeiger

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138 pages
Physiological, molecular and biochemical characterization of rodent extraocular muscles: Implications for Duchenne Muscular Dystrophy I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Ulrike Zeiger geboren am 11. März 1977 in Erlangen Greifswald, 04. Mai 2011 Dekan: Prof. Dr. Klaus Fesser 1. Gutachter: Prof. Dr. H. Brinkmeier (Institut für Pathophysiologie, Universität Greifswald) 2. Gutachter: Prof. Dr. R. Wiesner (Institut für vegetative Physiologie, Universität Köln) Tag der Promotion: 4. Oktober 20112 Table of Contents TABLE OF CONTENTS ABBREVIATIONS .................................................................................................................. 7 1. INTRODUCTION.............................................................................................................. 11 1.1 The Extraocular Muscles (EOMs) .......................................................... 11 1.2 Duchenne muscular dystrophy (DMD) .......................................................................... 18 1.2.1 Clinical & molecular background of DMD ..........................18 1.2.2 Pathogenesis of DMD .................................................................................................
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Physiological, molecular and biochemical
characterization of rodent extraocular muscles:
Implications for Duchenne Muscular Dystrophy

I n a u g u r a l d i s s e r t a t i o n
zur
Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
der
Mathematisch-Naturwissenschaftlichen Fakultät
der
Ernst-Moritz-Arndt-Universität Greifswald

vorgelegt von
Ulrike Zeiger
geboren am 11. März 1977
in Erlangen
Greifswald, 04. Mai 2011



























Dekan: Prof. Dr. Klaus Fesser

1. Gutachter: Prof. Dr. H. Brinkmeier (Institut für Pathophysiologie, Universität Greifswald)
2. Gutachter: Prof. Dr. R. Wiesner (Institut für vegetative Physiologie, Universität Köln)

Tag der Promotion: 4. Oktober 2011
2
Table of Contents
TABLE OF CONTENTS
ABBREVIATIONS .................................................................................................................. 7 
1. INTRODUCTION.............................................................................................................. 11 
1.1 The Extraocular Muscles (EOMs) .......................................................... 11 
1.2 Duchenne muscular dystrophy (DMD) .......................................................................... 18 
1.2.1 Clinical & molecular background of DMD ..........................18 
1.2.2 Pathogenesis of DMD ................................................................................................. 20 
1.4 Calcium homeostasis in normal and dystrophic muscle ...................... 23 
1.4.1 Excitation-contraction (E-C) coupling .........................................................23 
1.4.2 Store-operated calcium entry: role of TRP channels, Orai and STIM ........................ 25 
1.4.2.1 The TRP superfamily of cation channels ...............................................25 
1.4.2.2 Orai and STIM proteins .............................................................. 28 
1.4.3 Dysregulated calcium homeostasis in DMD ............................................................... 29 
1.3 Potential sparing mechanisms in EOM.................................................. 31 
1.4 Aim of the study ....................................................................................... 34 
2. MATERIALS AND METHODS ...................................................................................... 35 
2.1 Materials ................................................................................................... 35 
2.1.1 Chemicals .................................................................................................................... 35 
2.1.2 Reagents .............................................................................................. 36 
2.1.2 Consumables ............................................................................................................... 38 
2.1.3 Equipment & Devices ......................................................................... 39 
2.1.4 Animals ....................................................................................................................... 40 
2.1.5 Solutions and buffers for SDS-Page and Western blotting ......................................... 40 
2.1.7 Solutions for immunohisto- and cytochemistry ............................................42 
2.1.8 Primary antibodies ............................................................................. 43 
2.1.9 Secondary antibodies and nucleic acid (nuclear) stain ............................................... 43 
2.1.10 Solutions for cell culture of primary myoblasts ..................43 
2.1.11 Solutions for calcium imaging .................................................................................. 44 
3
Table of Contents
2.1.12 Molecular biology kits & reagents ........................................................................... 45 
2.1.12.1 Molecular biology kits ................................................................ 45 
2.1.12.2 Reverse Transcription (RT) reaction mixes ................45 
2.1.12.3 Primers and Probes ............................................................................................ 47 
2.1.12.4 SYBR Green reaction ................................................................ 49 
2.1.12.5 TaqMan reaction ..................................................................................49 
2.1.13 Software and Databases .................................................................... 53 
2.2 Methods ..................................................................................................... 54 
2.2.1 Tissue dissection ......................................................................................................... 54 
2.2.2 Tissue preservation ..................................................................................................... 55 
2.2.3 Western blotting .................................................................................. 55 
2.2.3.1 Sample preparation for Western blotting ............................................................. 56 
2.2.3.2 Determination of protein concentration ............................................................... 56 
2.2.3.3 SDS Page electrophoresis .................................................................................... 57 
2.2.3.4 Protein transfer (blotting) .................................................58 
2.2.3.5 Protein detection .................................................................................................. 59 
2.2.3.6 Stripping of blot membranes ..................60 
2.2.3.7 Protein quantification using densitometry ........................................................... 61 
2.2.4 Immunohisto- and cytochemistry .........................................61 
2.2.4.1 Preparation of cryosections ......................................................... 61 
2.2.4.2 Staining of cryosections .........................................................................61 
2.2.4.3 Staining of cultured myoblasts and myotubes ..................................................... 62 
2.2.4.4 Mounting of slides ............................................................................................... 62 
2.2.5 Cell culture .................................................................................................................. 63 
2.2.5.1 Acid washed cover slips ...................................................................................... 63 
2.2.5.2 Collagen I-coated cell culture dishes ........................................... 63 
2.2.5.3 Isolation of myoblasts ...................................................63 
2.2.5.4 Culture of primary myoblasts ....................................................... 64 
2.2.5 Intracellular calcium measurements (calcium imaging) ..................... 64 
2.2.6.1 Calcium imaging in myotubes ...............................................................64 
2.2.6.2 Analysis of calcium imaging experiments ................................... 65 
4
Table of Contents
2.2.7 RNA isolation and quantification ............................................................................... 65 
2.2.8 Reverse transcription .................................................................................................. 66 
2.2.9 Quantitative PCR (qPCR) ................................................................... 67 
2.2.9.1 SYBR Green based qPCR ......................................................................68 
2.2.9.2 Primer design for SYBR Green qPCR ................................................................. 68 
2.2.9.3 SYBR Green qPCR analysis: deltadelta Ct-method ......69 
2.2.9.4 TaqMan Assay based qPCR ................................................................................ 69 
2.2.9.5 TaqMan Assay analysis: absolute quantification ................................................. 70 
2.2.10 Statistical analyses .................................................................................................... 70 
3. RESULTS ................................................................................................... 71 
3.1 Calcium handling properties in rat EOM ..................................................................... 71 
3.1.1 Expression of myogenic markers in EOM myotubes ................................................. 71 
3.1.2 Calcium buffering in EOM and TA myotubes .............................................73 
3.1.3 mRNA expression levels of calcium handling proteins in EOM ................................ 76 
3.1.4 Protein expression of calcium handling proteins in EOM ............................79 
3.1.5 Immunohistochemistry of calcium handling proteins ................................................. 83 
3.2 Molecular components of the calcium homeostasis in normal and mdx mouse EOM
.................................................................................................................................................. 85 
3.2.1 Protein expression of calcium handling proteins ........................................................ 85 
3.2.2 Expression of TRP channel proteins in EOMs .....................88 
3.2.2.1 mRNA expression of TRPC channels ................................................................. 88 
3.2.2.2 Protein expression of TRPC1 and TRPC3 .....................89 
3.2.2.3 mRNA expression of TRPV channels ................................................................. 90 
3.2.2.4 Protein expression of TRPV4 ...................................................... 90 
3.2.2.5 mRNA expression of TRPM channels ................................................................. 91 
3.2.3 Expression of Orai1 and STIM1 ...................................................................93 
3.2.3.1 mRNA expression of Orai1 and Stim1 ....................................... 93 
3.2.3.2 Protein expression of Orai1 and STIM1 ........................93 
4. DISCUSSION ............................................................................................. 95 
4.1 Calcium buffering in rat EOM ....................................................................................... 95 
5
Table of Contents
4.2 Calcium buffering in mouse EOM ................................................................................. 99 
4.3 Model of the superior calcium homeostasis in EOM ......................... 100 
4.3 Expression of TRP channels, Orai and STIM in normal mouse muscle including
EOM ...................................................................................................................................... 101 
4.4 Expression of calcium handling proteins and channels in mdx muscle: Implications
for DMD ................................................................................................................................ 107 
4.5 Therapeutic interventions targeting calcium homeostasis in DMD .......................... 111 
5. SUMMARY ...................................................................................................................... 114 
6. REFERENCES ................................................................................................................. 116 
ACKNOWLEDGEMENTS ................................................. 138 


6
Abbreviations
Abbreviations

~ about
# number
10 x 10 times concentrated solution
1 x normal strength solution
ATPase adenosine triphosphatase
AU arbitrary units
Bp base pair
2+Ca calcium
CaCl calcium chloride 2
CALM calmodulin
2+CAMKII Ca /calmodulin dependent kinase II
CASQ calsequestrin
Cat# catalog number
cDNA complementary DNA
DHPR dihydropyridine receptor
DI water deionized water
DMEM Dulbecco’s Modified Eagle Medium
DNA desoxyribonucleic acid
DTT dithiotreitol
E-C excitation-contraction
ECL enhanced chemiluminsecence
EDTA ethylenediaminetetraacetic acid
EGTA ethylene glycol tetraacetic acid
EOM extraocular muscle
7
Abbreviations
ER endoplasmatic reticulum
FBS fetal bovine serum
FGF fibroblast growth factor
FW forward
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
h hour
H O water 2
HCl hydro chloric acid
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HRP horse radish peroxidase
IgG immunoglobulin G
kDa kiloDalton
M Molar (mol/l)
mg milligram
µg microgram
µl microliter
µM micromolar (µmol/l)
Milli-Q water ultrapure laboratory grade water
min minute
miRNA microRNA
ml milliliter
mm millimeter
mM millimolar (mmol/l)
mRNA messenger RNA
+Na sodium
NaCl sodium chloride
8
Abbreviations
nM nanomolar (nmol/l)
NCX1/Slc8a1 sodium-calcium exchanger
nm nanometer
NTC non-template control
ºC degrees Celsius
oN over night
PARV parvalbumin
PBS phosphate buffered saline
Pen/Strep Penicillin/Streptomycin
PKA protein kinase A
PLN phospholamban
2+PMCA plasmamembrane Ca ATPase
PVDF polyvinylidene fluoride
qPCR quantitative polymerase chain reaction
RGN regucalcin
RNA ribonucleic acid
ROS reactive oxygen species
rpm revolutions per minute
RT reverse transcription
RV reverse
RYR ryanodine receptor
s second or seconds
SD standard deviation
SEM standard error of the mean
SDS sodium dodecyl sulfate
2+SERCA sarcoplasmic reticulum Ca ATPase
9
Abbreviations
SLN sarcolipin
SR sarcoplasmic reticulum
SRL sarcalumenin
STIM stromal interaction molecule
Std standard
2+SOCE store-operated Ca entry
TA tibialis anterior
TBS Tris buffered saline
TBST Tris buffered saline Tween
temp. temperature
TIF(F) tagged image file format
TNNC troponin C
TRP transient receptor potential
10