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

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 component