Investigating TNF inhibition of IGF-1 signalling via …...Investigating TNF inhibition of IGF-1...

Investigating TNF inhibition of IGF-1

signalling via JNK in cell culture models of

skeletal muscle atrophy

Bijanka L. Gebski

This thesis is presented for the degree of Doctor of Philosophy

School of Anatomy and Human Biology

The University of Western Australia

2009

ii

Dedication

This thesis is dedicated to the wonderful women in my family, especially my mother.

You are all so very different, yet have met life’s challenges with enormous strength and

dignity. The determination to rise above any challenge, and the attitude of ‘becoming

stronger’ as a result of the journey are the most powerful qualities one may endeavour

to behold. I will be forever grateful for your guidance and inspiration.

iii

Abstract

The pro-inflammatory cytokine tumour necrosis factor (TNF) has a critical role in

skeletal muscle atrophy. The catabolic effect of TNF is partially due to abrogation of the

anabolic insulin-like growth factor 1 (IGF-1) signalling pathway. However, the precise

signalling events that lead to the loss of myofibrillar protein following activation of

TNF receptor are unknown.

The over arching aim of the study is to determine the mechanisms of by which TNF

induces atrophy in differentiated muscles cells. To achieve this aim a series of

experiments were performed to: 1) investigate the molecular events that lead to TNF

mediated myofibre atrophy, 2) determine to what extent c-Jun N-terminal Kinase (JNK)

signalling plays a part in TNF induced myotube atrophy, and in TNF-mediated

inhibition of IGF-1 induced hypertrophy, and 3) use inhibitors of JNK to block the

catabolic effects of TNF.

1) To investigate the molecular events that lead to TNF mediated myofibre atrophy, the

experiments were conducted using C2C12 mouse myotube cultures and primary

myotube cultures derived from FVB mice, and transgenic mice which over-express

Class 2 IGF-1 Ea in skeletal muscles (IGF:C2). The treatment of mature C2C12 and

FVB primary myotubes (respectively at 7 and 4 days after fusion medium) with 10

ng/mL of TNF for 3 days resulted in statistically significant myotube atrophy

(decreased mean width). The observed TNF-mediated atrophy has not previously been

demonstrated in tissue cultured myotubes. In contrast, addition of IGF-1 (20 ng/ml) to 7

day C2C12 myotubes for 3 days resulted in significant hypertrophy. Where exogenous

combinations of TNF and IGF-1 were added to the C2C12 myotubes, the atrophic effect

of TNF was prevented (and conversely the hypertrophic effect of IGF-1 was prevented).

Furthermore, when TNF was added to the IGF:C2 myotube cultures, there was no

statistically significant difference between the mean size of the TNF treated myotubes,

compared to the untreated IGF-1:C2 myotubes. These results taken into consideration

demonstrate the protective effect of IGF-1 against TNF induced atrophy.

iv

2) To assess the role of JNK signalling in TNF induced myotube atrophy, it was first

necessary to determine if TNF induced JNK phosphorylation in day 7 C2C12 myotubes.

Signalling studies confirmed that treatment with TNF (20 ng/ml) for 10 min was

sufficient to induce JNK phosphorylation in the C2C12 myotubes. Three JNK inhibitors

(2 ATP-competitive; SP600125 and AS601245 and 1 ATP-non-competitive; TAT-

TIJIP) were assessed for biological toxicity in C2C12 myotubes. The most suitable

inhibitor was TAT-TIJIP and was thus used in subsequent studies. Inhibition of JNK

activity by TAT-TIJIP was confirmed indirectly by detecting nuclear translocation of c-

Jun, which is a downstream target of phosphorylated JNK. Immunohistochemical

analyses showed nuclear localisation and phosphorylation of c-Jun in TNF treated

myotubes. Nuclear localisation and phosphorylation of c-Jun was not observed in

cultures pre-treated with TAT-TIJIP before TNF treatment, nor in the untreated control

myotubes.

3) The use of JNK inhibitors to block the catabolic effects of TNF was tested using

C2C12 and primary myotube cultures. Pre-treatment of C2C12 and primary FVB

myotubes with the JNK inhibitor TAT-TIJIP, 30 min before TNF administration (for 3

days) prevented myotube atrophy. The mean width of myotubes pre-treated with TAT-

TIJIP prior to TNF treatment closely resembled that of the control myotubes.

Administration of TNF in combination with TAT-TIJIP for 3 days to C2C12 myotubes

prevented myotube atrophy and unexpectedly resulted in hypertrophy when compared

to the mean widths of untreated and TAT-TIJIP treated myotubes. This trend was also

demonstrated in the FVB primary cultures. These combined results strongly support the

role of JNK in TNF-mediated atrophy. Preliminary studies were carried out in vivo

using the mdx mouse model of muscular dystrophy, TAT-TIJIP was administered via

intraperitoneal injection to the mice for 3 days at a dose of 10 mg/ml, however the

results form this study are inconclusive.

These novel observations are of considerable interest to the field of muscle wasting

because they demonstrate for the first time TNF-mediated myotube atrophy, the role of

JNK in situations of TNF induced muscle atrophy, and explore the use of JNK

inhibitors to prevent muscle atrophy.

v

Acknowledgments

I would like to acknowledge and thank the following people for their contributions:

Professional

I would like to thank my supervisors Professor Miranda Grounds and Dr Thea

Shavlakadze from the School of Anatomy and Human Biology, UWA for their interest

in my work and with assisting me in compiling this thesis. I also acknowledge my

external supervisor Dr Marie Bogoyevitch; formerly from the School of Biomedical,

Biomolecular & Chemical Sciences, UWA but now located at Department of

Biochemistry and Molecular Biology, University of Melbourne, Victoria.

I would also like to thank Dr Jon Whitehead (Head, Cell Signalling Group, Diamantina

Institute for Cancer, Immunology and Metabolic Medicine, Princess Alexandra

Hospital, University of Queensland) for taking the time to help me optimise IRS-1 assay

and for allowing me to undertake supervised experiments at your laboratory.

This research was made possible by generous funding from the National Health and

Medical Research Council of Australia and a Postgraduate scholarship from UWA.

Personal

Completing this thesis would not have been anywhere near possible if it was not for the

continuous support from:

My amazing (and very entertaining) family; especially Mum and Dad for believing in

me, loving me, supporting me for so very long and never giving up on the idea that B

will one day finish! Dan for your continuous, continuous and then some more support

when I needed it the most. You are an amazingly strong person, I don’t know how you

do it, and I admire you for that, thank you Frank. My grandmothers: Baba for gluten

free meals on wheels and Nanna for the stories and favoured envelopes of cash. Aunty

vi

Barb for ever and always the ‘you can do it be’ emails, Optus ‘20 minute free time’

counselling sessions, couture inspired gifts, fashion and trash mags (!) and for being my

#1 fashion spend supporter (now what can I blame when I buy that ludicrously over

priced item, PhD is done!). Uncle Alan, for as materialistic and self absorbed as it

sounds (Gen Y, Al), the ‘therapeutics’ (please never leave Bacardi Lion!!). Aunty Don

for being Aunty Don - knowing that there was always a place to go when I needed to

‘unload’ and be fed at the same time was so very important to my mental and physical

well being. Uncle Greg, thanks for getting great jobs so that I had a place to stay in

Melbourne and the home-away-from-home, Mt Lawley residence.

ALL my friends!! You guys are ‘A’! The girls (Liv, Nat, Lozza, Mallis, Donna,

Chantelle); thanks so much for sticking by and continually asking me to ‘come out b’

when you knew too well that I had ‘uni on’ of had ‘to do my thesis’. Microbio friends;

Carmel and Janie - we all (eventually) made it out onto the other side, and for that we

are tops! Chantelle, deli days made me strong, but the ability to digest more gluten than

ever is all thanks to you. Thea Shav, thank you. When I took over your seat in room

2.36 I had big shoes to fill. Thanks for sharing many laughs (and tears) with me over

numerous ‘nosh-ski’ dinners, ‘muddies’ and for good times NYC. You are so strong

shav, thanks for making me ‘get on with it’ when I really, really didn’t want to

anymore. You are a ‘tops’ friend. Girls - you are all very special to me and I am very

lucky to have wonderful friends like you.

Room 2.36; what an experience that was. I could never have dreamt that by enrolling in

a PhD I would also be sharing an office with an amazing group of hilariously funny,

intelligent, witty and determined people, with whom I would form incredibly strong

friendships. Thanks guys. The ‘ghetto’ party crew of 2008 (well wicked) and not to

forget ‘tissue couture good times’. Special thanks to Marg for getting me thru the

toughest times of my PhD, you were my ‘mum’ away from home.

Jason; thank you for providing the inspiration I so desperately needed through all things

art, music, anime, Hayao Miyazaki, Murakami and the beautiful books. If it wasn’t for

you I would not have stayed at the job for any longer than 2 months and would have had

to leave 18/33 Third Ave.

vii

My Iaido Sensei Jason Anstey, thank you for guiding me while I travel on my path of

understanding the complexity of the Japanese sword and the concept of budo.

And a big thank you to Talijancich Winery; Tali family, you are amazing! Thank you

for being such wonderful friends (and employers!) and for producing my most favourite

wine, the ‘Talijancich Viognier’. To my new colleagues at PwC; thanks so much for

understanding that I had to go home (and not Bar One) to write this thesis, and here it

is! ☺

viii

Declaration

I hereby declare that the work contained within this thesis is entirely my own.

___________________________

Bijanka Gebski

May 2009

ix

Table of Contents

Title ................................................................................................................................. i

Abstract ......................................................................................................................... ii

Acknowledgements ................................................................................................... iv

Declaration ................................................................................................................... v

Table of Contents ...................................................................................................... vi

List of Figures............................................................................................................xii

List of Tables ............................................................................................................. xv

Abbreviations ........................................................................................................... xvi

Chapter One: Introduction Overview .............................................................................................................. 1

1.1 Skeletal muscle .............................................................................................. 2

1.2 Physiological and pathological remodelling of skeletal muscle ................ 3

1.2.1 Skeletal muscle hypertrophy ............................................................ 3

1.2.2 Skeletal muscle atrophy ................................................................... 3

1.3 Duchenne Muscular Dystrophy ................................................................... 5

1.3.1 The molecular basis of Duchenne Muscular Dystrophy .................. 5

1.3.2 Mdx mouse model of DMD ............................................................. 6

1.3.2.1 Mdx mouse, muscle necrosis and voluntary exercise ....... 7

1.4 Insulin like Growth Factor -1 (IGF-1) ....................................................... 8

1.4.1 Protein analogues of IGF-1: naturally occurring and synthetic ...... 9

1.4.1.1 Naturally occurring IGF-1 isoforms ................................. 9

1.4.1.2 Naturally occurring analogue of IGF-1: Des(1-3)IGF-1... 9

1.4.1.3 Synthetic analogue of IGF-1: LONGTMR3IGF-1 ............. 9

1.4.2 IGF-1 signalling in skeletal muscle cells ....................................... 10

1.4.3 The protective benefits of IGF-1 for muscle wasting conditions:

IGF-1 over expressing animal model...................................................... 13

1.5 Tumour Necrosis Factor (TNF) ................................................................ 13

1.5.1 TNF exacerbates muscle damage................................................... 16

x

1.5.2 TNF activates c-Jun N-terminal Kinase (JNK).............................. 17

1.5.3 IGF-1 and TNF cross talk: involvement of JNK............................ 18

1.5.4 Inhibitors of JNK Kinases.............................................................. 21

1.5.4.1 ATP-competitive ............................................................. 21

1.5.4.2 ATP-non-competitive...................................................... 22

Aims and Hypothesis of the Thesis .................................................................... 23 Overall Aim ....................................................................................................... 23

Specific Aims...................................................................................................... 23

Hypothesis .......................................................................................................... 23

Chapter Two: Materials and Methods ............................................................. 24 2.1 Cell Culture ................................................................................................. 24

2.1.1 Immortalised muscle cell lines....................................................... 24

2.1.1.1 C2C12 murine myotube cultures .................................... 24

2.1.1.2 L6 rat myotubes cultures................................................. 24

2.1.2 Animal strains ................................................................................ 24

2.1.2.1 Animals used for primary muscle cell culture

preparations ................................................................................. 25

2.1.2.2 Animals used to test the effects of the JNK inhibitory

peptide TAT-TIJIP ...................................................................... 25

2.1.3 General cell culture conditions....................................................... 25

2.1.3.1 Preparation of primary cell cultures................................ 25

2.1.3.2 Resurrection of cyropreserved cell stocks....................... 26

2.1.3.3 Trypsinising and seeding sells onto plates ...................... 27

2.2 Reagents and Antibodies ............................................................................ 29

2.2.1 Treatment reagents ......................................................................... 29

2.2.2 JNK Inhibitory peptides ................................................................. 29

2.3 Antibodies .................................................................................................... 30

2.3.1 Western blotting ............................................................................. 30

2.3.2 Immunohistochemistry................................................................... 30

2.4. Myotube morphology studies .................................................................... 30

2.4.1 Treatment regime for myotube cultures......................................... 30

xi

2.4.2 Immunohistochemistry................................................................... 31

2.4.3 Image analysis: measuring myotube widths .................................. 31

2.4.4 Statistical analysis .......................................................................... 33

2.5 Western blot quantitation of JNK phosphorylation in Day 7 C2C12

myotubes ............................................................................................................ 33

2.5.1 Treatment regime for western blot analysis of phospho-JNK ....... 33

2.5.2 Extraction and sample preparation................................................. 34

2.5.3 Protein quantitation ........................................................................ 34

2.5.4 Protein Sample Preparation............................................................ 35

2.5.4.1Whole cell lysates ............................................................ 35

2.5.4.2 Immunoprecipitation of IRS-1 ........................................ 35

2.5.5 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) .............. 35

2.5.6 Protein transfer for western blotting .............................................. 37

2.5.7 Blocking non-specific antibody binding ........................................ 37

2.5.8 Protein detection ............................................................................ 38

2.5.9 Chemiluminescent signal detection to observe protein.................. 38

2.5.10 Densitomerty for AKT signalling study....................................... 38

2.6 Animal studies to test the effects of TAT-TIJIP....................................... 38

2.6.1 Exercise induced muscle damage in adult mdx mice..................... 38

2.6.2 Animal anaesthesia and sacrifice ................................................... 39

2.6.3 Tissue sampling and processing..................................................... 39

2.6.4 Haematoxylin and eosin staining ................................................... 40

2.6.5 Image analysis: quantification of necrosis ..................................... 40

2.6.6 Statistical analysis .......................................................................... 41

Chapter Three: Validation of cell culture models of IGF-1 induced

skeletal muscle hypertrophy ................................................................................ 42

Introduction ............................................................................................................... 42

Results .......................................................................................................................... 43 3.1 Establishing optimum culture and treatment Conditions....................... 43

3.1.1 Comparing C2C12 myotube formation on different substrates ..... 43

3.1.2 Determining the optimum fusion medium and seeding density

xii

for C2C12 myotube formation ................................................................ 44 3.2 Preliminary Studies to Determine the Biological Activity and Optimum

Dosage of IGF-1 Required for Inducing Hypertrophy in C2C12 and L6

Myotube Cultures (passage > 25) .................................................................... 46

3.2.1 Effects of IGF-1 (LONGTMR3IGF-I) treatment on day 3 mouse

C2C12 muscle myotubes (passage > 25) ................................................ 46

3.2.2 Effects of Des(1-3)IGF-1 treatment on day 3 mouse C2C12

muscle myotubes (passage > 25) ............................................................ 47

3.3 Effects of IGF-1 treatment on day 3 rat L6 myotubes (passage > 25).... 49

Discussion.................................................................................................................... 52

Chapter Four: Morphological effects of IGF-1 and TNF treatment on

cultured myotubes.................................................................................................... 56

Introduction ............................................................................................................... 56

Results .......................................................................................................................... 58 4.1 Effects of IGF-1 treatment on day 7 mouse C2C12 myotubes................ 58

4.2 The effect of TNF treatment on day 7 mouse C2C12 myotubes ........... 58

4.2.1 Independent C2C12 myoblast fusion assay to test the biological

activity of TNF........................................................................................ 58

4.2.2 Effects of TNF treatment on day 7 mouse C2C12 myotubes ........ 59

4.3 Effects of combined treatment with IGF-1 and TNF on day 7 mouse

C2C12 muscle myotubes................................................................................... 61

4.4 Effects of TNF on control FVB and primary cultures of transgenic

IGF:C2 mouse myotubes .................................................................................. 62

4.4.1 Effect of TNF on day 4 FVB primary mouse myotubes................ 62

4.4.2 Testing the effect of TNF on transgenic IGF-1 class 2 primary

muscle cells ............................................................................................. 63

Discussion.................................................................................................................... 65 IGF induced myotube hypertrophy................................................................. 66

TNF induced myotube atrophy........................................................................ 66

xiii

Chapter Five: The role of TNF activated JNK in muscle wasting in

tissue cultured myotubes ....................................................................................... 71

Introduction ............................................................................................................... 71 Results .......................................................................................................................... 73

5.1 TNF activates the JNK signalling pathway in C2C12 myotubes ............ 73

5.1.1 Optimising conditions for detecting TNF activation of the JNK

signalling pathway in day 7 C2C12 myotubes........................................ 73

5.1.2 Titration study to determine if JNK phosphorylation is dependant

on dosage of TNF.................................................................................... 75

5.2 JNK inhibitors used in TNF induced myotube atrophy studies ............. 76

5.2.1 Selection of a suitable JNK inhibitor: evaluation of three

different inhibitors at two dosage levels in C2C12 myotubes ................ 76

5.2.2 Experiments to determine if TNF induced myotube atrophy

can be prevented by pre-treatment with the JNK inhibitor TAT-TIJIP 79

5.2.2.1 Analysis of the morphological effects of TAT-TIJIP

on untreated and TNF treated C2C12 myotubes......................... 79


on untreated and TNF treated control FVB mouse myotubes

in primary cultures ...................................................................... 80


on untreated and TNF treated transgenic IGF:C2 mouse

myotubes in primary cultures...................................................... 81

5.3 Confirmation of the inhibition of JNK phosphorylation by

TAT-TIJIP: immunofluorescent nuclear localisation of the

transcription factor c-Jun................................................................................. 83

5.3.1 Time course analysis of the nuclear localisation of

phosphorylated c-Jun in day 7 C2C12 myotubes.................................... 84

5.3.2 Immunofluorescent microscopy analysis of TAT-TIJIP

inhibition of c-Jun nuclear localisation (phosphorylation) in TNF

induced myotube atrophy in C2C12, FVB and IGF:C2 cultures ............ 86

xiv

5.4 Analysis of IRS-1 and AKT (downstream of IGF-1) to further

investigate TNF inhibition of IGF-1 signalling via JNK, in the model of

C2C12 myotube atrophy .................................................................................. 88

5.4.1 Optimising western blotting conditions for immunoprecipitated

IRS-1 from C2C12 Day 7 myotubes treated with IGF-1 ........................ 88

5.4.2 Western blotting analysis of AKT from C2C12 Day 7 myotubes

treated with IGF-1 or TNF ...................................................................... 90

Discussion.................................................................................................................... 92

Chapter Six: Preliminary study to investigate the effects of

TAT-TIJIP on reducing exercised induced myofibre necrosis

in vivo in mdx mice .................................................................................................. 96

Introduction ............................................................................................................... 96

Results .......................................................................................................................... 98 6.1 The effect of JNK inhibitor TAT-TIJIP on reducing myofibre necrosis

in 6 week old voluntary exercised mdx mice .................................................. 98

6.1.1 Distance run by the mdx mice........................................................ 98

6.1.2 Histological analysis of the effects of TAT-TIJIP on reducing

myofibre necrosis in voluntary exercised mdx mice .............................. 99

Discussion.................................................................................................................. 101

Chapter Seven: General Discussion ................................................................ 104

Overview.................................................................................................................... 104

7.1 Establishment of myotube culture models of IGF-1 induced

hypertrophy and TNF mediated atrophy ..................................................... 104

7.2 Combination treatments to investigate the effects of IGF-1 and

TNF................................................................................................................... 105

7.3 Involvement of JNK in TNF mediated myotube atrophy and use

of JNK inhibitors............................................................................................. 106

7.1.4 In vivo studies: preliminary findings .................................................... 108

References ................................................................................................................. 109

xv

List of Figures

Figure 1.1 Skeletal Muscle Structure .................................................................. 2

Figure 1.2 Intracellular signalling pathways initiated downstream of IGF-1

receptor............................................................................................... 12

Figure 1.3 Intracellular signalling pathways initiated downstream of the TNF

receptor 15

Figure 1.4 Schematic representation of the events occurring following

sarcolemmal damage in dystrophic myofibres................................... 16

Figure 1.5a Damaging effects of TNF in skeletal muscle is proposed to be

mediated by JNK inhibition of IGF-1 signalling ............................... 20

Figure 1.5b Potential use of JNK inhibitors for preventing JNK mediated

down regulation of IGF-1 signalling in skeletal muscle .................... 20

Figure 2.1 Neubauer haemocytometer................................................................. 28

Figure 2.2 Measuring myotube widths using Image Pro Plus software .............. 33

Figure 2.3 Illustration of the Western blotting transfer cassette.......................... 37

Figure 2.4 Exercise equipment for voluntary running......................................... 39

Figure 2.5 Typical histology of dystrophic mdx quadriceps muscle................... 41

Figure 3.1 Myotube formation on different surface coatings.............................. 44

Figure 3.2 The fusion of C2C12 myoblasts into myotubes in two media

(DMEM +/- HS) at two different initial plating densities in 35mm

collagen (type I) coated dishes........................................................... 45

Figure 3.3 Effects of IGF-1 treatment on C2C12 myotubes .............................. 48

Figure 3.4 Effects of Des(1-3) IGF-1 treatment on C2C12 myotubes ............... 49

Figure 3.5 Effects of IGF-1 treatment on L6 myotubes ..................................... 50

Figure 3.6 Immunostained L6 myotubes treated with IGF-1 ............................. 51

Figure 4.1 Fusion assay to test biological activity of TNF ................................. 59

Figure 4.2 Immunofluorescent microscopy images of Day 10 C2C12

myotubes treated with IGF-1 or TNF for 3 days ............................... 60

Figure 4.3 Quantitative analysis of mean myotube widths of untreated

(control), IGF-1 and TNF treated C2C12 cultures ............................ 61

Figure 4.4 Quantitative analysis of myotube widths of untreated (control), 62

xvi

IGF-1, TNF and combined IGF-1 and TNF treated C2C12

cultures ..............................................................................................

Figure 4.5 Quantitative analysis of myotube widths from TNF atrophy

experiments conducted on Wild type FVB and Transgenic

IGF:C2 primary skeletal muscle cell cultures ................................... 63

Figure 5.1 Optimising conditions for detecting TNF phosphorylation of JNK

in differentiated (day 7) C2C12 myotubes: preliminary

experiments ....................................................................................... 74

Figure 5.2 Time course assay of TNF phosphorylation of JNK in

differentiated (day 7) C2C12 myotubes: final protocol .................... 75

Figure 5.3 Titration study to determine the minimum effective dose of TNF

to induce phosphorylation of JNK in differentiated (day 7) C2C12

myotubes ........................................................................................... 76

Figure 5.4 Toxicity evaluation of three JNK inhibitors on day 10 C2C12

myotubes ........................................................................................... 78

Figure 5.5 Mean width of untreated control C2C12 myotubes and C2C12

myotubes treated with TNF alone, TAT-TIJIP alone, combination

of TAT-TIJIP and TNF, TAT alone and combination of TAT and

TNF ................................................................................................... 80

Figure 5.6 Mean width of untreated control primary FVB myotubes and

FVB myotubes treated with TNF alone, TAT-TIJIP alone,

combination of TAT-TIJIP and TNF, TAT alone and combination

of TAT and TNF ............................................................................... 81

Figure 5.7 Mean width of untreated control primary IGF:C2 myotubes and

IGF:C2 myotubes treated with TNF alone, TAT-TIJIP alone,

combination of TAT-TIJIP and TNF, TAT alone and combination

of TAT and TNF ............................................................................... 83

Figure 5.8 Immunofluorescence microscopy images depicting the nuclear

localisation of phosphorylated c-Jun in day 7 C2C12 myotubes

treated with TNF ............................................................................... 85

Figure 5.9 Immunofluorescent microscopy images depicting reduced nuclear

localisation of phosphorylated c-Jun in muscle cell cultures pre

treated with TAT-TIJIP inhibitor 1 hr before treatment with TNF ... 87

xvii

Figure 5.10 Comparison of tyrosine phosphorylation (pY) of IRS-1 in Insulin

stimulated C2C12 cells from two different laboratories ................... 90

xviii

Figure 5.11 Activation of AKT in differentiated (day 7) C2C12 myotubes

treated with TNF or IGF-1 ................................................................

91

Figure 6.1 Percentage of myofibre necrosis in right quadriceps muscle of

untreated and TAT-TIJIP treated voluntary exercised adult mdx

mice ................................................................................................... 100

xix

List of Tables

Table 2.1 Dimensions of various tissue culture plates and the myoblast

plating densities used in these studies .................................................. 29

Table 2.2 Concentration of BSA in the protein assay standards .......................... 34

Table 2.3a Composition of Resolving gel: 12% polyacrylamide in 1 M Tris-

HCL pH 8.8 .......................................................................................... 36

Table 2.3b Composition of Stacking gel: 6% polyacrylamide in 0.5 M Tris-

HCL pH 6.8 .......................................................................................... 36

Table 6.1 The relation between the voluntary distance ran to the amount of

exercised induce myofibre necrosis .................................................... 98

xx

Abbreviations

AKT Serine/threonine protein kinase

ANOVA Analysis of variance

ATP Adenosine triphosphate

bFGF Basic fibroblast growth factor

BSA Bovine serum albumin

CSA Cross sectional area

DEX Dexamethasome

DMD Duchenne Muscular Dystrophy

DMEM Dulbecco’s Modified Eagle’s Medium

DMSO Dimethyl sulfoxide

ERK Extra cellular signal related kinase

EDTA Ethylenediaminetetraacetic acid

FCS Foetal calf serum

FM Fusion Media

Foxo Forkhead box, sub-group O transcription factor

GH Growth hormone

GM Growth media

H&E Haematoxylin and eosin

HS Horse Serum

IFN-γ Interferon-γ

IGF-1 Insulin like growth factor-1

IGF-2 Insulin like growth factor-2

IGF-1R Insulin like growth factor-1 receptor

IGF-2R Insulin like growth factor-2 receptor

IGFBP Insulin like growth factor binding proteins

IL Interleukin

IRS-1 Insulin receptor substrate-1

JIP JNK-interacting protein

JNK c-Jun N-terminal Kinase

MAFbx Muscle atrophy F box or Atrogin-1

xxi

MAPK Mitogen-activated protein kinases

mIGF-1 Skeletal muscle-specific exon-1 Ea isoform of IGF-1

mTOR mammalian target of rapamycin

NFĸβ Nuclear factor ĸβ

PBS Phosphate buffered saline

PDL Poly-D-Lysine

PI3K Phosphatidylinositiol 3-kinase

PVA Polyvinyl alcohol mounting medium

PVDF Polyvenylidene difluoride

ROS Reactive Oxygen Species

RT Room temperature

SDS Sodium Dodecyl Sulfate

sTNF Soluble TNF

TA Tibialis anterior

TBS Tris buffered saline

TIJIP Truncated Inhibitor of JNK Interacting Protein

TIM TNF receptor associated factor-interacting motifs

TNF Tumour necrosis factor

TNF-R1 TNF receptor 1

TNF-R2 TNF receptor 2

TRAF TNF receptor associated factor

TRIS Tris(hydroxymethyl)aminomethane

UT Untreated

xxii

Publications in Referred Journals

Grounds, M. D., Radley, H. G., Gebski, B. G., Bogoyevitch, M. A. and Shavlakadze, T.

(2008). "Implications Of Cross-Talk Between Tumour Necrosis Factor And Insulin-

Like Growth Factor-1 Signalling In Skeletal Muscle." Clin Exp Pharmacol Physiol.

Manuscripts in Preparation

c-Jun N-terminal Kinases (JNKs) are critical mediators of TNF-induced myotube

atrophy

Gebski BLa, Bogoyevitch M.A.b, Grounds M.D. a, Shavlakadze T.S. a

a School of Anatomy and Human Biology, the University of Western Australia

b Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and

Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Victoria,

3010, AUSTRALIA.

Chapter One Introduction

1

Chapter One

Introduction

Overview

This following is a comprehensive review of the background literature pertinent to the

present study. The chapter opens with a general discussion on skeletal muscle biology

and the importance of skeletal muscle hypertrophy and atrophy is introduced. The

molecular basis of Duchenne Muscular Dystrophy (DMD) and the mdx animal model of

DMD are then discussed in relation to the prominent muscle necrosis associated with

voluntary exercise. The relationship between DMD and the c-Jun N-terminal Kinase

(JNK) is briefly discussed before an in-depth review of both the Insulin like growth

factor-1 (IGF-1) and Tumour necrosis factor (TNF) signalling pathways. The

involvement of JNK in the cross-talk between these two pathways is demonstrated and

the significant importance of this kinase in mediating the deleterious effects of TNF in

situations of muscle inflammation is discussed. Finally, various inhibitors that aim to

block the actions of activated JNK are reviewed with the advantages and disadvantages

of each presented.


2

1.1 Skeletal muscle

Skeletal muscles make up approximately 40% of the mass of the human body and are

composed of multinucleated muscle myofibres (or myotubes) which are formed by the

fusion of myoblasts (muscle cells). The myofibres are filled with highly organised

contractile proteins and arranged in bundles within several different layers of fibrous

connective tissue and are attached to skeletal bones by connective tissue and tendons

(Figure 1.1). Skeletal muscles are under voluntary nervous control (somatic nerve

stimulation); their primary function is movement (contraction) in addition to

maintaining normal body positioning and posture (Saladin 2004).

Figure 1.1: Skeletal Muscle Structure. (A) Skeletal muscle is arranged in bundles. The bundles are encased within three sheaths of connective tissue; epimysium covers the whole muscle, perimysium covers the bundles of the myofibre and the endomysium covers each individual myofibre (SEER, n.d) (B) Myofibres are composed of sarcoplasm filled with striated muscle cells (myofibrils) and are encased within the sarcolemma (membrane). Figure adapted from Bannister et al. (1995).

A

Sarcolemma (membrane)

Peripheral Nucleus

Mitochondria

Myofibril (muscle cell)

Sarcoplasmic Reticulum

Transverse Tubules

B


3

1.2 Physiological and pathological remodelling of skeletal muscle

1.2.1 Skeletal muscle hypertrophy

Skeletal muscle hypertrophy is an increase in the size, diameter (cross sectional area,

CSA) and mass of individual myofibres. In adults this develops as a compensatory

response to resistance exercise, increased physiological loading or enhanced nutritional

uptake in vivo (Grounds 2002; Jackman and Kandarian 2004). Skeletal muscle adapts to

loading changes by increasing the size and amount of contractile proteins within the

sarcomeres of each myofibre. This results in an increase in the CSA of myofibres, their

associated force production, and also an alteration in protein synthesis and degradation

rates (Goldspink 1991; Adams and Haddad 1996; Russell et al. 2000).

Numerous factors and signals also trigger hypertrophy of skeletal muscles, for example

it is well known that Insulin-like growth factor-1 (IGF-1 - which is a subject of study in

this thesis), hormones, cytokines and anabolic steroids all have a crucial role in inducing

skeletal muscle hypertrophy (Semsarian et al. 1999) (Boonyarom and Inui 2006). The

important intracellular signalling pathways responsible for skeletal muscle hypertrophy

are discussed in greater detail in Section 1.4 of this thesis.

1.2.2 Skeletal muscle atrophy and cachexia

Skeletal muscle atrophy is a reduction in the CSA and mass of individual myofibres,

accompanied by a decrease in myofibrillar protein content, decreased force production,

and lower fatigue resistance (Boonyarom and Inui 2006). The biochemical and

enzymatic responses include the reduced capacity to synthesize new protein and the up-

regulation of pathways leading to increased protein breakdown (Jackman and Kandarian

2004; Shavlakadze and Grounds 2006).

Many factors can result in muscle atrophy including neuromuscular disorders,

immobilisation/muscle disuse, denervation (Shavlakadze and Grounds 2006),

inflammatory conditions associated with cancer (Baracos et al. 1995), sepsis (Fang et al.

2000), Acquired Immune Deficiency Syndrome (AIDS) (Gonzalez-Cadavid et al. 1998),

burn injury, glucorticocoid treatment (Argiles et al. 2007), diabetes (Price et al. 1996)

and ageing and starvation (Mitch and Goldberg 1996).


4

As the pathological aetiology of muscle atrophy vary (Shavlakadze and Grounds 2006),

it is important to distinguish muscle atrophy resulting from disuse/denervation or

starvation, from muscle wasting induced in pathological inflammatory conditions as a

result of high levels of pro-inflammatory cytokines (also known as cachexia), such as

tumour necrosis factor (TNF) (Argiles et al. 2001). At a recent symposium, the

definition of cachexia was refined, the general consensus of the group was that

cachexia ‘is a complex metabolic syndrome’ resulting from an adaption to an

underlying illness (such as cancer) and is associated with anorexia (loss of appetite),

inflammation, insulin resistance, anaemia and increased muscle protein breakdown

(Evans et al. 2008). Due to the central role of inflammation (specifically the elevated

levels of inflammatory cytokines including TNF) in cachexia and other situations of

atrophy, therapies targeting the inflammatory pathways will continue to attract

considerable interest to reduce the severity of muscle wasting (Evans et al. 2008).

A common characteristic of atrophying muscle is induction of the ubiquitin-proteasome

protein degradation pathway (Lecker et al. 2004). Two important genes (atrogenes) up-

regulated in atrophic states are ubiquitin ligase Muscle atrophy F box (MAFbx or

Atrogin-1) and Muscle RING finger-1 (MuRF-1) (Lecker et al. 2004; Latres et al.

2005). Another important signalling pathway associated with regulation of muscle mass

is that of IGF-1; loss of IGF-1 signalling may be a key factor for skeletal muscle

atrophy (Grounds 2002).

Conversely, it is now widely understood that muscle mass can be regulated by important

growth factors (such as IGF-1) and that the loss of IGF-1 and its downstream signalling

events may be key factors in accelerating skeletal muscle atrophy (Grounds 2002).

Recent interesting reports have suggested that down-regulation of IGF-1 activated

signalling events through TNF-stimulated activation of a family of cytosolic stress-

activated kinases, known as the c-Jun N-terminal Kinase (JNKs). The action of active

JNK on key proteins involved in IGF-1 signalling pathways can inhibit IGF-1 signalling

and therefore prevent the positive effects of IGF-1 (Broussard et al. 2003; Broussard et

al. 2004; Strle et al. 2006). This is discussed in greater detail in Section 1.5 (see below).

The balance between TNF and IGF-1 signalling is also implicated in the extensive

muscle wasting associated with the lethal muscle disease Duchenne Muscular


5

Dystrophy (DMD). As such, and animal models of this disease (such as the mdx mouse)

are of considerable interest and importance for the development of new therapeutic

targets related to these interacting signalling pathways.

1.3 Duchenne Muscular Dystrophy

1.3.1 The molecular basis of Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy is a lethal X-linked muscle wasting disorder arising

from mutations within the dystrophin gene (Hoffman et al. 1987). These mutations

result in the absence of functional dystrophin, leading to fragility of the sarcolemma and

can result in myofibre necrosis (Blake et al. 2002). Without prenatal screening and

genetic counselling, DMD occurs at a frequency of 1 in 3500 live male births (Emery

1991). The disease is clinically evident in early childhood, usually between 3 to 5 years

of age (Emery 2002). Due to continual muscle wasting, boys with DMD are restricted to

a wheel chair by 12 years of age and 90% die in their late teens or early twenties,

usually due to cardiac or respiratory failure (Blake et al. 2002). However, the use of

mechanical assisted ventilation has increased the lifespan of DMD patients to the mid

20s (Bach et al. 1997).

Following the discovery of the molecular basis of DMD in 1987, research has been

directed towards the development of new disease treatments based on this newly

acquired and improved understanding of the underlying causes of the muscle

dysfunction. Gene replacement strategies, including myoblast and stem cell transfer,

have had limited success in the mouse and human trials (Grounds and Davies 2007;

Davies and Grounds 2007). Alternative forms of molecular therapy have also been

considered. These include the delivery of genes or minigenes via viral or non-viral

vectors (Odom et al. 2007; Wang et al. 2009), utrophin up regulation (Li et al. 2009),

gene correction (Bertoni et al. 2005; Maguire et al. 2009) and the use of antisense

oligonucleotides to mask mutations within the dystrophin gene (Wu et al. 2008;

Heemskerk et al. 2009; Yokota et al. 2009). To date, there is still no effective treatment

for DMD. The current research project thus focuses on a new strategy related to the role

of inflammation in the breakdown of dystrophic muscle.


6

1.3.2 Mdx mouse model of DMD

The understanding of DMD was considerably enhanced following the discovery of the

mdx mouse as a reliable animal model. Bulfield and colleagues showed that a

spontaneous X-chromosome linked mutation had arisen in their inbred C57BL/10

colony of mice; the serum of these mutant mice contained high levels of muscle-derived

pyruvate kinase and creatine kinase, which is also present in DMD (Bulfield et al.

1984). The mice also exhibited histological lesions similar to those characteristic of

early DMD, involving necrosis of muscle fibres (myofibril necrosis) (Bulfield et al.

1984; Partridge 1991). This research group named the mice ‘mdx’ (Bulfield et al. 1984).

The molecular basis of the dystrophy presented in the mdx mouse was characterised in

1989. This was achieved by cloning complementary DNA (cDNA) of mouse

dystrophin, with the sequence being used in a polymerase chain reaction (PCR) to

amplify both the normal and the mdx dystrophin transcripts (Sicinski et al. 1989).

Sicinski's group determined that the mdx mouse arose from a point mutation in exon 23

of the dystrophin gene. This single base change within exon 23 results in only 27% of

the dystrophin protein being translated prior to encountering the premature stop codon

(Sicinski et al. 1989).

One of the most significant factors of the mutation in the mdx mouse is that it does not

result in the large fibrotic lesions characteristic of human DMD. Furthermore, and most

importantly, the mice appear to be able to regenerate their skeletal muscle. Certain

muscles (such as the diaphragm) do display some of the features seen in human DMD

patients (O'Brien and Kunkel, 2001), but there has been speculation in regards to the

genetic homology of the mdx mice and that observed in human DMD (Partridge 1991;

Blake et al. 2002). In the attempt to produce an animal model that would more closely

reflect human DMD, several mouse strains mdx2Cv, mdx3Cv, mdx4Cv and mdx5Cv

were generated by the deliberate induction of ethyl-nitroso-urea at the mdx locus

(Chapman et al. 1989; O'Brien and Kunkel 2001; Blake et al. 2002). However, despite

different mutations in the dystrophin gene, these mice appeared phenotypically similar

to the naturally occurring mdx model (Partridge 1991; Blake et al. 2002) and thus the

mdx mouse is continued to be used.


7

1.3.2.1 Mdx mouse, muscle necrosis and voluntary exercise

Although a functional form of the dystrophin protein is absent in mdx mice (and they

are thus susceptible to muscle necrosis), the mutation does not result in the same

severity of pathology, specifically the eventual replacement of muscle by fibrous fatty

connective tissue as observed in human DMD boys (Gillis 1999). The marked

differences in the severity of the dystropathology between humans and mice may be

largely accounted for by large differences in the size and growth parameters of the 2

species (Grounds et al. 2008). However, the mdx mouse remains the most convenient

and clinically relevant mammalian model to screen drugs for the potential clinical

treatment of DMD boys (Granchelli et al. 2000; Payne et al. 2006; Grounds et al. 2008).

The age at which the mdx mice are used for muscle necrosis studies is critically

important. Histological examination of muscle sampled from mdx mice younger than 3

weeks of age bears little resemblance to that associated with the dystrophic phenotype,

but by around day 21, there is an acute onset of muscle damage in the limb and

paraspinal muscles (Coulton et al. 1988; McGeachie et al. 1993; Grounds and Torrisi

2004; Shavlakadze et al. 2004). It has been reported that the percentage of muscle

necrosis in the tibialis anterior muscle increases to approximately 20-40% in 3 week old

mdx mice (Shavlakadze et al. 2004). This rapid, acute onset of myofibre damage is then

followed by intensive muscle regeneration, which stabilises at about 6 weeks of age to a

relatively low level of around 6% (McGeachie et al. 1993).

As this reduced and ongoing low level of muscle necrosis presents a difficult model to

assess the effectiveness of drug intervention to reduce myofibre necrosis, mice have

been subjected to voluntary exercise to increase muscle damage and necrosis (Archer et

al. 2006; Hodgetts et al. 2006; Radley and Grounds 2006). This allows for potential

therapies (such as the interventions described in Chapter 6) to be more effectively tested

in adult mdx mice (Grounds et al. 2008).

The interventions to be evaluated in mdx mice that are of central interest to the present

research relate to modulating the effects of IGF-1 and TNF. The key features of IGF-1

and TNF are therefore discussed in more detail (see below).


8

1.4 Insulin like growth factor -1 (IGF-1)

The Insulin-like Growth Factor (IGF) system consists of three peptide hormones/ligands

(Insulin, IGF-1 and IGF-2), their receptors (insulin receptors, IGF-1 type 1 receptor;

IGF-1R and IGF-2 type 2 receptor; IGF-2R) and six IGF binding proteins (IGFBP-

1,2,3,4,5,6) (Hwa et al. 1999). The insulin receptor is a main mediator of the biological

functions of insulin, while the IGF-1 receptor mainly mediates cellular responses of

IGF-1 and IGF-2. It is considered that the IGF-2 receptor does not have a major role for

IGF-1 and IGF-2 signal transductions, but it is responsible for binding and thus

reducing IGF-2 levels during foetal development (Le Roith et al. 2001).

IGF-1 is a potent growth factor in vitro and in vivo, and its actions are regulated by

interactions with IGFBPs (Rutanen and Pekonen 1990). IGF-1 is essential for normal

muscle growth during development, stimulating proliferation and differentiation in

myoblasts (Engert et al. 1996), exerting anabolic effects such as myofibre hypertrophy

(increased size and protein content) (see below), muscle regeneration in adults and the

prevention of atrophy (muscle wasting) in ageing muscle (Musaro et al. 2001; Grounds

2002; Glass 2005). In addition IGF-1 induces satellite cell proliferation and

differentiation and interleukin 4 (IL-4); a myoblast recruitment factor for fusion with

existing myotubes (Horsley et al. 2003).

Growth hormone (GH) induces the expression of IGF-1 in many tissues, such as the

liver, pancreas, muscles, intestines, kidney, brain and adipose tissue, and therefore IGF-

1 acts as the main mediator of GH effects (Le Roith et al. 2001). IGF-1 is produced by a

range of tissues (Froesch et al. 1985), however the liver is the primary production site

(approximately 75%) of circulating IGF-1; the balance is produced locally by other

tissues. The IGF-1 gene gives rise to multiple isoforms of pre-processed IGF-1 (which

differ by termination peptides) and can exert different biological functions (Shavlakadze

et al. 2005).


9

1.4.1 Protein analogues of IGF-1: naturally occurring and synthetic

1.4.1.1 Naturally occurring IGF-1 isoforms

The mammalian IGF-1 gene gives rises to several isoforms of pre-processed IGF-1

which differ by termination peptides and can exert different biological functions. The

processed form is a 70-amino acid long (molecular weight 7649 Da) single IGF-1

polypeptide cross-linked by three disulfide bridges (Rinderknecht and Humbel 1978). In

mammalian species, the structure of IGF-1 is highly conserved, with mouse IGF-1 (Bell

et al. 1986) differing to human IGF-1 (Rinderknecht and Humbel 1978) by only four

amino acids. For a more detailed report on the complexity of IGF-1 isoforms and the

structure of rodent IGF-1 see (Shavlakadze et al. 2005).

1.4.1.2 Naturally occurring analogue of IGF-1: Des(1-3)IGF-1

Des(1-3)IGF-1 is a naturally occurring truncated analogue of IGF-1. Des(1-3)IGF-1 is a

67 amino acid protein that differs to human and bovine IGF-1 only by the omission of

the tripeptide Gly-Pro-Gly from the N-terminus (Ballard et al. 1987). Des(1-3)IGF-1

was discovered as the growth promoting activity of bovine colostrum on L6 myoblasts

(Francis et al. 1986). Since Des(1-3)IGF-1 has only 1% of the affinity for IGFBPs

(which normally inhibit the biological actions of naturally occurring IGF-1) (Ross et al.

1989), Des(1-3)IGF-1 has been reported to have approximately 10 times the potency of

naturally occurring IGF-1 with both cell hypertrophy and proliferation induced at

substantially lower concentration than that of IGF-1 in many cell types (Francis et al.

1988; Carlsson-Skwirut et al. 1989).

The synthesis of Des(1-3)IGF-1 is believed to occur though post-translational

modification of mature IGF-1, following the removal of the leader sequence and the C-

terminus extension (Ballard et al. 1987) It has been suggested that the N-terminus

extension sequences (encoded by exons 1 and 2) directs the specificity of a signal

peptidase to yield either IGF-1 of Des(1-3)IGF-1 (Ballard et al. 1996).

1.4.1.3 Synthetic analogue of IGF-1: LONGTMR3IGF-1

Long R3 Insulin-like Growth Factor-I (LONGTMR3IGF-1) is an 83 amino acid analogue

of IGF-I comprising the complete IGF-I sequence with the substitution of glutamic acid

(E) at position 3 with arginine (hence R3), and a 13 amino acid extension peptide at the


10

N-terminus. It is produced in Escherichia coli as a single, non-glycosylated, polypeptide

with a molecular weight of 9111 Da (Francis et al. 1992).

LONGTMR3IGF-1 is significantly more potent than both naturally occurring IGF-1 and

the IGF-1 analogue of Des(1-3)IGF-1 in vitro and is characterised by a reduced affinity

(greater then 1000-fold) for binding to IGFBPs (Francis et al. 1992). This significantly

reduced affinity is the result of the 13 amino acid extension. When tested in an L6

myoblast protein assay, LONGTMR3IGF-1 was the most potent peptide in stimulating

protein synthesis in the myoblasts, with Des(1-3)IGF-1 and then IGF-1 being the least

potent in that order (Francis et al. 1992). Studies performed in embryonic kidney

(HEK293) cells corroborate these finding; Voorhamme & Yandel (2006) reported that

in serum free cultures, LONGTMR3IGF-1 was a more potent growth and survival factor,

than either insulin or IGF-I .

The studies in this thesis exclusively use LONGTMR3IGF-1 to capitalise on its potency

in stimulating IGF-receptors and subsequent signalling events. Therefore, from Chapter

3, and continuing for the remainder of the thesis, whenever the term ‘IGF-1’ is used, it

refers to the LONGTMR3IGF-1 isoform.

1.4.2 IGF-1 signalling in skeletal muscle cells

It is well documented that IGF-1 plays a crucial role in mediating the anabolic

regulation of muscle mass (Florini et al. 1996) and can induce hypertrophy (Section

1.2.1) through either autocrine or paracrine mechanisms (DeVol et al. 1990). IGF-1

stimulates the proliferation and differentiation of myoblasts (Engert et al. 1996), induces

the uptake of amino acids and incorporation into proteins, regulation of glucose uptake,

and suppression of protein degradation (Florini and Magri 1989; Florini et al. 1996).

Numerous mouse muscle cell culture studies have demonstrated that treatment of

multinucleated muscle cells (myotubes) with the IGF-1 analogue LONGTMR3IGF-1

induced hypertrophy of myotubes (Florini et al. 1996; Rommel et al. 1999; Rommel et

al. 2001). Similarly, studies using the L6E9 (a subclone of the L6 cell line) show IGF-1

induced hypertrophy in myotubes via the transfection of a muscle specific IGF-1 vector

that is only activated during cell differentiation (Musaro and Rosenthal 1999; Musaro et

al. 1999; Fanzani et al. 2006). It has also been reported that the same signalling


11

pathways which mediate myotube hypertrophy in the C2C12 cells are also upregulated

when IGF-1 was administered to L6 myotubes (Li et al. 2005). Furthermore, the ability

of IGF-1 over-expression to reduce denervation atrophy was demonstrated in skeletal

muscle in vivo (Stitt et al. 2004; Shavlakadze et al. 2005) and appeared to be due to

suppression of the up-regulation of atrophy associated genes (Stitt et al. 2004; Latres et

al. 2005). All these functions are mediated through IGF-1 binding to its receptor (IGF-

1R) a ligand-activated receptor tyrosine kinase.

IGF-1R is a trans-membrane protein consisting of two extracellular ligand binding α-

subunits (which contain the IGF-1 binding site) and two β-subunits (Figure 1.2). When

IGF-1 binds to the extracellular α-subunits, specific tyrosine kinases in the receptor β-

subunits undergo autophosphorylation (activation) (Jacobs et al. 1983; Rubin et al.

1983; Ullrich et al. 1986). An early molecule involved in linking the receptor kinase to

the biological actions of IGF-1 is tyrosine phosphorylation of insulin receptor substrate -

1 (IRS-1) (Figure 1.2), a 135 kDa tyrosine phosphoprotein and SH2 domain containing

protein ((Keller et al. 1993; Rothenberg et al. 1991). IRS-1 serves as docking protein for

other cytoplasmic signalling molecules that contain SH2 domains, including

phosphatidylinositiol (PI) 3-kinase and Grb2 (Florini et al. 1996). Activated PI3K then

phosphorylates membrane phospholipid phosphatidylinositol 4,5-bisphosphate to

produce phosphatidylinositol 3,4,5-trisphosphate (Matsui et al. 2003) (Figure 1.2). This

modified lipid is recognised by the serine/theronine kinase AKT (Alessi et al. 1997),

which when at the membrane is phosphorylated and activated by PDK (PDK-1)

(Vivanco and Sawyers 2002) (Figure 1.2).


12

Figure 1.2: Intracellular signalling pathways initiated downstream of IGF-1 receptor.

In skeletal muscle cells, the pathway downstream of PI3K has been linked to

differentiation, survival, hypertrophic growth and the prevention of muscle atrophy

(Rommel et al. 1999). The activation of the defined PI3K/AKT pathway in C2C12

cultured myotubes has been described and defined a as crucial pathway for regulating

protein synthesis in skeletal muscle cells and muscle hypertrophy (Glass 2003; Rommel

et al. 2001; Latres et al. 2005). Increase phosphorylation and activation of AKT has

been demonstrated in cultured muscle cell models of IGF-1 induced hypertrophy

(Rommel et al. 2001) and in in vivo models of functional overload induced hypertrophy

(Bodine et al. 2001).

Similarly, as AKT is activated by the IGF-1 receptor, so are direct and indirect targets

of AKT; mammalian target of rapamycin (mTOR), p70S6K and 4EBP-1, all key

regulatory protein involved in protein synthesis (Rommel et al. 2001; Latres et al.

2005). The significance of the PI3KAKT/mTOR pathway in mediating hypertrophy in

skeletal muscle cells was demonstrated using the mTOR inhibitor, rapamycin. When

administered in vivo rapamycin almost completely prevented hypertrophic growth of

skeletal muscle (Bodine et al. 2001) and blocked all but 1% of the gene activated by

IGF-through the PI3K/AKT pathway (Latres et al. 2005).


13

Activation of the PI3K/AKT pathway can also reduce myofibre atrophy and counteract

protein degradation by inversely regulating a number of atrophy related genes such as

MAFbx (Latres et al. 2005). The down regulation of the PI3K/AKT pathway results in

the upregulation of atrophy related genes (see Section 1.2.2) via the desphohorylation of

Forkhead box, sub-group O transcription factors (Foxo) and their consequent

translocation into the nucleus (Sandri et al. 2004; Latres et al. 2005). It is Foxo3 that

directly activates transcription from MAFbx promoter (in vivo and cell culture) and

induce muscle atrophy (Sandri et al. 2004), however AKT phosphorylates Foxo

transcription factors on various sites, which results in the Foxo protein being unable to

translocate to the nucleus and thus their transcriptional functions are inhibited (Brunet et

al. 1999).

1.4.3. The protective benefits of IGF-1 for muscle wasting conditions: IGF-1 over

expressing animal model

In situations of muscle wasting, such as dystrophy, the protective benefits of IGF-1 have

been investigated and discussed in depth in various reviews (Shavlakadze et al. 2005;

Grounds et al. 2008). The over-expression of skeletal muscle specific IGF-1 (mIGF-1)

in dystrophic mdx mice can significantly reduce the severity of the dystrophic

pathology, most likely due to reduced muscle necrosis and increased protein synthesis

in the muscles (Shavlakadze et al. 2004). Over-expression of IGF-1 was generated in

transgenic mdx/mIGF-1 mice using a tissue-restricted transgene which encoded the

skeletal muscle-specific exon-1 Ea isoform of IGF-1 (called mIGF-1) (Musaro et al.

2001). At one year of age, these mice showed a 40% increase in muscle mass when

compared to mdx mice (Barton et al. 2002). Further to these observations, transgenic

mdx/mIGF-1 mice exhibited a significant reduction in the amount of fibrosis normally

observed in the diaphragms of mdx mice (Barton et al. 2002).

1.5 Tumour Necrosis Factor (TNF)

Tumour necrosis factor (TNF) is a pro-inflammatory cytokine that is elevated during

muscle wasting (cachetic) conditions such as cancer, AIDS and sepsis (Argiles et al.

2001). Initially it was thought that TNF was released by bacteria, and that TNF

mediated endotoxin induced the necrosis of certain tumours in mice, resulting in a


14

muscle-wasting syndrome (cachexia) in tumour-bearing mice (Carswell et al. 1975).

Later it was agreed that TNF and endotoxin were separate factors, because TNF has a

direct cytotoxic effect on various tumour cells in vitro (Hoffmann et al. 1978; Old

1981). It was also found that TNF and the protein cachetin are identical, with the latter

also involved in muscle wastage associated with cancer (Beutler et al. 1985).

It is well documented that TNF has a critical role in skeletal muscle atrophy. TNF is

also the principal cytokine mediating cachexia (muscle wasting) (Argiles et al. 1997;

Glass 2005; Arthur et al. 2008)., TNF, however also has a pivotal role in initiating a

broad range of diverse cellular responses, including inhibition of skeletal muscle

proliferation and differentiation (Miller et al. 1988; Szalay et al. 1997; Meadows et al.

2000; Foulstone et al. 2001; Coletti et al. 2002; Foulstone et al. 2004), growth arrest

(Darzynkiewicz et al. 1984), inflammatory effects (Beutler and Cerami 1988; Peterson

et al. 2006), mediation of the immune response, and apoptotic and necrotic cell death

mechanisms (Laster et al. 1988; Denecker et al. 2001; Tolosa et al. 2005). Furthermore,

it has been reported that expression levels of TNF are elevated in conditions associated

with muscle pathology such as inflammatory myopathy (Lundberg et al. 1995; Saito et

al. 2000; Efthimiou 2006), DMD (Tews 2005) and cancer cachexia (Tracey et al. 1988;

Argiles et al. 1997; Pajak et al. 2008).

TNF and the other members of the TNF ligand family stimulate a complex array of post

receptor signalling events through binding to specific receptors. The cellular signalling

response to TNF seemingly depends on the cell type and physiological conditions. Of

the approximate 29 receptors that make up the TNF receptor (TNF-R) family, two are of

significant importance to TNF exerting its biological functions, these being TNF

receptor type 1 (TNF-R1) and TNF receptor type 2 (TNF-R2). TNF-R1 is the primary

receptor for soluble TNF (sTNF) and TNF-R2 the main receptor for membrane

integrated TNF (memTNF) (Wajant et al. 2003).

TNF-R1 contains a death domain (DD) that is activated upon ligand (TNF) binding,

which then activates the caspase cascade and induces apoptosis and necrosis (Laster et

al. 1988; Denecker et al. 2001; Tolosa et al. 2005). TNF-R2 is distinct to TNF-R1

because TNF-R2 contains TRAF (TNF receptor associated factor)-interacting motifs

(TIM), which upon ligand binding, cause the recruitment of TRAF family members


15

(Dempsey et al. 2003). Members of the TNF-R family activate the mitogen-activted

protein kinases (MAPK) which regulate various cellular activities, such as gene

expression, mitosis, differentiation, and cell survival/apoptosis (Pearson et al. 2001),

PI3K, p38, extra cellular signal regulated kinase (ERK), nuclear factor ĸB (NF- ĸB)

(closely associated with inflammation), capsases (involved in Apotosis) and, of

considerable relevance to the present study, c-Jun-N-terminal kinase (JNK) (Karin and

Gallagher 2009). The role of TNF varies markedly according to the cell type (Shutze et

al. 1992; Wajant et al. 2003) thus in this study it will be looked at specifically from the

role it plays in skeletal muscle cells.

Figure 1.3: Intracellular signalling pathways initiated downstream of the TNF receptor. Binding of TNF to its receptor stimulates at least four distinct signal transduction pathways (Wajant et al. 2003). These are (1) apoptosis via interaction of the TNF-receptor complex and the Fas-associated protein (FA) with death domain (DD), (2) activation of JNK (associated with stress related cellular responses and atrophy), (3) activation of p44/p42MAPK (Erk 1/2) (associated with atrophy) and (4) activation of receptor-interacting protein which then activates p38 MAPK, which is responsible for activiating atrophy related genes (Li et al. 2005) and is also involved in myogenesis in skeletal muscle (Cuenda and Cohen 1999; Chen et al. 2007), and NF-κB (associated with protein loss) (Reid and Li 2001; Wajant et al. 2003). Figure modified from Sethi et al. (2008).


16

1.5.1 TNF exacerbates muscle damage

TNF is expressed by a wide range of inflammatory cells and elevated in inflammatory

myopathies and DMD (Kuru et al. 2003; Saito et al. 2000). TNF is also produced by

adipose tissue (Coppack 2001; Weisberg et al. 2003) which is often pronounced in

DMD muscles. In normal muscle, some small lesions of the sarcolemma may be

repaired, and myofibre necrosis does not occur (Bansal et al. 2003). However, initial

muscle lesions may also be exacerbated by pro-inflammatory cytokines (such as TNF)

and inflammatory cells (Tidball 2005) leading to necrosis and muscle breakdown

(Figure 1.3). The blockade of TNF (by either Remicade or Enbrel) in vivo has been

reported to protect dystrophic muscle from necrosis in the mdx mouse model of DMD

(Grounds and Torrisi 2004; Grounds et al. 2005). Both these drugs are currently in wide

clinical use to treat inflammatory disorders such as arthritis or Crohn’s disease.

Figure 1.4: Schematic representation of the events occurring following sarcolemmal damage in dystrophic myofibres. Lack of functional dystrophin (A) makes the myofibre membrane susceptible to damage (B). Where TNF levels are low, such tears may be rapidly resealed (C) by fusion of the membrane vesicles to the edges of damaged sarcolemma, whereby the myofibre returns to a relatively intact state (D). Alternatively (E) if TNF levels are elevated, the membrane fails to be repaired and necrosis of the sarcoplasm takes place. Inflammatory cells rapidly invade the necrotic area and start to remove damaged tissue. Necrosis is followed by regeneration (F), in which muscle precursor cells proliferate, differentiate and fuse together to form myotubes and replace the necrotic muscle segment. It is proposed that the cytokine TNF can promote necrosis in preference to resealing. Adapted from Shavlakadze et al. (2004) and Hodgetts et al. (2006).

TNF

Basement membrane Sarcolemma Satellite cell

A. Dystrophic Myofibre

B. Damage to sarcolemma

TNF

F. RegenerationE. Necrosis

C. Resealing

Sarcoplasm

D. “Intact” myofibre


17

1.5.2. TNF activates c-Jun N-terminal Kinase (JNK)

In major inflammatory conditions, elevated levels of the key pro-inflammatory

cytokine, TNF results in a significant increase in skeletal muscle protein degradation

(Tisdale 2005). In addition, the stress-activated c-Jun N-terminal kinases (JNKs) have

also been suggested to be involved in the pathophysiology of major inflammatory

conditions (Strle et al. 2006) such as inflammatory bowel disease (Roy et al. 2008),

mouse models of DMD (Kolodziejczyk et al. 2001; Lang et al. 2004) and in rat models

of rheumatoid arthritis (Han et al. 2001).

JNKs are a subfamily of threonine/serine (Ser/Thr) mitogen-activated protein kinases

(MAPKs) which are activated in response to a number of stimuli, including pro-

inflammatory cytokines, stress stimuli such as UV irradiation, a range of growth factors

(Kyriakis and Avruch 2001). There are three JNK encoding genes and at least 10 splice

variants (Gupta et al. 1996). The products of JNK1 and JNK2 are ubiquitously

expressed, whereas JNK3 expression is generally considered to be restricted to the

central nervous system (Yang et al. 1997; Kumagae et al. 1999).

The JNKs act within a protein kinase cascade, which when stimulated by extracellular

stimuli (stress, UV, cytokines) or intracellular stimuli (ER stress). The JNKs undergo

dual phosphorylation by the MAPK kinases MKK4 and MKK7 (Butterfield et al. 1997;

Barr and Bogoyevitch 2001). As emphasised by the name, JNKs are able to

phosphorylated and activate the transcription factor c-Jun (Cheng and Feldman 1998)

via the phosphorylation of Serine 63 (Ser63) and Serine 73 (Ser73) in the N-terminus of

c-Jun (Derijard et al. 1994; Kyriakis and Avruch 2001). The binding of JNKs to the

amino-terminal region of c-Jun results in nuclear localisation and phosphorylation of

this transcription factor (Derijard et al. 1994) leading to the activation of a number of c-

Jun gene dependent events such as apoptotic cell death (Tibbles et al. 1996; Tanaka and

Hanafusa 1998).

Activation of JNKs may contribute to progression of the disease pathogenesis of

muscular dystrophy because the mdx mouse displays muscle specific activation of

JNK1 (Kolodziejczyk et al. 2001). However, the levels of phosphorylated JNKs in

dystrophic muscle alter depending on the muscle type (Kolodziejczyk et al. 2001). In

gastroconemius muscle from both 6 week old mdx and control normal mice, low levels


18

of phosphorylated JNK1 were detected in both strains, whereas there was a significant

difference in levels of activated JNK2 in mdx compared to controls (Nakamura et al.

2005). Previously, JNK1 expression was studied in 12 month old mdx and the

mdx:MyoD-/- mice. It was found that there was a marginal increase in phosphorylated

JNK1 in mdx hindlimb muscle lysates compared to normal controls, with a substantial

increase in activated JNK1 in the mdx:MyoD-/- lystates, compared to both the mdx and

wild type strains (Kolodziejczyk et al. 2001). However, analysis of mdx diaphragm

muscle showed increased phosphorylated JNK 1 compared to that observed in the wild

type. It has been recently reported that increased levels of phosphorylated JNK1 were

present in the diaphragms of 7 week old mdx mice when compared to age matched

C57BL/10 controls (Hnia et al. 2007). Alterations in the expression and/or

phosphorylation levels in the different dystrophic muscles are not limited to JNK1 and

2. It has been reported that in mdx muscle, the activation levels of ERK1/2 (p44/p42

MAPK), p70 S6 kinase (p70s6k) and p38 kinase are also dependant on the muscle type

(Lang et al. 2004).

It is also important to note that there are potential benefits of TNF relevant to muscle

regeneration during physiological conditions. A study by Chen et al (2007) showed that

C2C12 that were placed in low-serum differentiation medium release low amount of

TNF. The study also reported that upon addition of a TNF neutralizing antibody to the

differentiation medium, p38 activation was blocked and differentiation suppressed,

suggesting that TNF regulates myogenesis and muscle regeneration via p38.

1.5.3 IGF-1 and TNF cross talk: involvement of JNK

While there is a general consensus that TNF plays a critical role in skeletal muscle

atrophy, the precise role of TNF activated JNK in situations of TNF mediated myofibre

atrophy is still largely unknown. There is now strong evidence that TNF can inhibit

IGF-1 expression (Frost et al. 2003) or abrogate IGF-1 receptor signalling via inhibition

of tyrosine phosphorylation of the IGF-1 docking protein, IRS-1 (Rui et al. 2001; Frost

et al. 2003; Broussard et al. 2004; Broussard et al. 2003; Strle et al. 2006; Grounds et al.

2008). It has been proposed that this inhibitory effect of TNF on IGF-1 signalling

appears to be mediated by JNK phosphorylation of IRS-1 (Broussard et al. 2003; Strle

et al. 2006) (Figure 1.4a).


19

Studies examining the role of JNK in signalling downstream of the insulin receptor

propose that JNK binds to, and phosphorylates, IRS-1 on Serine 307 (Ser307) (Aguirre et

al. 2000; Aguirre et al. 2002) or on Serine 318 (Ser318) (Mussig et al. 2005).

Phosphorylation of Ser307 and Ser318 induces a conformational change in the

phosphotyrosine binding domain of IRS-1, thus reducing its affinity for the insulin

receptor (Hiratani et al. 2005). IRS-1 is also considered to be a docking protein for the

IGF-1 receptor (Keller et al. 1993) (Figure 1.4a). Phosphorylation of IRS-1 on Ser307

downstream of the IGF-1 receptor results in inhibition of the PI3/AKT/mTOR pathway,

the predominant pathway downstream of IGF-1 signalling responsible for protein

synthesis (Rommel et al. 2001; Latres et al. 2005).

The role of JNK in mediating TNF induced inflammatory events and protein

degradation in skeletal muscle is only just beginning to be explored and much is still not

known. As such, the therapeutic potential of protein kinases are being increasing

targeted and investigated in the search for new therapies (Bogoyevitch et al. 2005). The

use of inhibitory peptides has allowed researchers to study the biological functions of

JNK in mammalian systems without the need for JNK gene knockout models. Of

particular interest to this study, a JIP-derived JNK inhibitor has been previously used to

establish that JNK mediates TNF suppression of IGF-1 in differentiating skeletal muscle

cells (Strle et al. 2006). Recently, the implications of TNF induced JNK activation in

skeletal muscles was also discussed, with the authors hypothesising that the inhibition

of JNK is beneficial for muscle wasting diseases such as DMD (Grounds et al. 2008).

Since it has been confirmed that JNK is a negative regulator of IGF-1 signalling in

C2C12 myoblasts (Strle et al. 2006), the next stage is to target JNK for inhibition in

C2C12 cell cultures models of TNF induced myotube atrophy (Fig .1.4b and see

Chapter 3).

Compared to other MAPKs pathways, the JNK pathway is defined by its interaction

with the JNK-interacting protein (JIP) family of scaffold proteins. Barr et al (Barr et al.

2002) demonstrated that an 11-mer peptide (TI-JIP) potentially inhibited JNK activity in

vitro. Through conjugation to the Tat peptide, it is rendered cell permeable and have

thus been used to investigate the investigate the effects of JNK in cells (Borsello et al.

2003; Kendrick et al. 2004; Kaneto et al. 2004). The specific cell permeable JNK

inhibitory peptides derived from JIP assessed in the present study (Section 1.5.4).


20

Figure 1.5a: Damaging effects of TNF in skeletal muscle is proposed to be mediated by JNK inhibition of IGF-1 signalling. In this image (A) TNF ligand induced activation of TNF-R results in the activation of c-Jun N Terminal kinase (JNK). (B) The damaging effects of TNF on skeletal muscle have been identified as to be mediated by JNK associating with IRS-1. Activated JNK phosphorylates IRS-1, which induces a conformational change and impairs the ability of IRS-1 to associate with IGF-1R resulting in the inhibition of IGF-1 signalling. (C) The absence of IGF-1 receptor binding to IRS-1 possibly results in a decrease in protein synthesis and an increase in protein degradation and muscle necrosis. Figure adapted from Grounds et al. (2008).

Figure 1.5b: Potential use of JNK inhibitors for preventing JNK mediated down regulation of IGF-1 signalling in skeletal muscle (A) TNF ligand induced activation of TNF-R results in the activation of c-Jun N Terminal kinase (JNK) (B) By targeting JNK for inactivation by specific JNK inhibitors, this counteracts the inhibitory action of JNK on IGF-1 signalling. (c) IGF-1 signalling is maintained which results in increase muscle protein synthesis and down regulation of muscle atrophy and necrosis. Figure adapted from Grounds et al. (2008).


21

1.5.4 Inhibitors of JNK kinases

The therapeutic potential of targeting protein kinases is a topic of increasing recent

interest; for comprehensive reviews see (Bogoyevitch et al. 2004; Bogoyevitch et al.

2005; Bogoyevitch and Arthur 2008). Studies involving determining the function of

JNK have largely depended on the use of blockers to inhibit JNK activity. These JNK

inhibitors have been categorised into two groups; chemical inhibitors that are ATP-

competitive (target the ATP binding site of JNK and subsequently other MAPK) and

newer peptide inhibitors that are ATP-non-competitive (directly bind to JNK)

(Bogoyevitch 2005).

1.5.4.1 ATP-competitive

ATP-competitive protein kinase inhibitors are peptides that compete for the ATP-

binding site of protein kinases and therefore inhibit protein kinase activity (Bogoyevitch

2005). Currently the most widely used inhibitor of the JNKs is SP600125

(anthra[1,9]pyrazole-6(2H)-one) (Bennett et al. 2001; Bogoyevitch 2005), although its

specificity has been questioned (Bain et al. 2003). However, when administered to

C2C12 myoblasts, SP600125 considerably attenuated the protein expression of the pro-

inflammatory cytokines TNF, IL-1 and LPS induced IL-6 (Frost et al. 2003). As it is

beyond the scope of this present study to review the structure, mode of activity and

therapeutic use of SP600125, the following reviews (Bogoyevitch 2005; Bogoyevitch

and Arthur 2008) should be consulted for a more detailed discussion.

AS601245 ((benzothiazol-2-yl) acetonitrile) is another new ATP competitive inhibitor

that is commercially available (Gaillard et al. 2005; Bogoyevitch and Arthur 2008).

AS601245 has shown to be beneficial in models of focal and global cerebral ischemia

(Carboni et al. 2004; Carboni et al. 2007). It has also been shown that treatment with

AS601245 resulted in a significant reduction on infarct size caused by myocardial

ischemia in anaesthetized rats (Ferrandi et al. 2004). Recently AS6001245 was also

successfully administered in a neonatal rat model of pneumococcal meningitis to

demonstrate that JNK may not be involved in pneumococcal meningitis-induced

hippocampal apoptosis (Sury et al. 2008).


22

1.5.4.2 ATP-non-competitive

ATP-non-competitive competitive protein kinases are peptides that do not compete for

the ATP-binding site of protein kinases, but through other mechanisms, such as

competing for binding to the peptide or protein binding site to inhibit protein-kinase

activity (Bogoyevitch 2005). The JIP derived peptides, such as TAT-TIJIP, TI-JIP,

JNKI-1 peptides (JIP-1-HIV-TAT, L-JNKI-1 and the protease-resistant D-amino acid

retroinverse peptide D-JNK1) have been shown to kinetically act in a substrate

competitive manner (Barr et al. 2004) and bind directly to the substrate docking domain

of JNK (Barr et al. 2004).

The cell permeable JNK inhibitors (TAT-TIJIP and D-JNKI-1) are composed of two

molecular structures. Both inhibitors contain a common cell permeable, 10 amino acid

human immunodeficiency virus-Tat cell transport sequence (TAT), that facilitates the

entry of the inhibitor into the target cells (Vives et al. 1997). The inhibitors are different

in their JNK binding sequences; TAT-TIJIP contains an 11 amino acid sequence

(Truncated Inhibitor of JNK Interacting Protein; TIJIP) (Kendrick et al. 2004) whereas

D-JNKI-1 contains a longer 20 amino acid sequence (JNK Binding Domain) (Borsello

et al. 2003).

The shorter of the two inhibitors (TAT-TIJIP) has been shown to have a beneficial

effect in preventing necrotic cell death in vitro (Arthur et al. 2007), whereas the longer

JNKI-1 peptides have been reported to be neuroprotective when tested in vivo in rat

models of cerebral ischemia (Borsello et al. 2003).

The protective effects of the ATP-competitive inhibitor SP600125, AS60125 and the

ATP-non-competitive inhibitor TAT-TIJIP in preventing TNF induced myotube atrophy

in cultured skeletal muscle cells Chapter 4 and 5 and in vivo in mdx mice models of

exercised induced skeletal muscle necrosis in Chapter 6.

Aims and Hypothesis

23

Aims and Hypothesis of the Thesis

Overall Aim

To determine the mechanisms by which TNF induces atrophy in differentiated skeletal

muscle cells.

Specific Aims

1. Validate, replicate and optimise a C2C12 muscle cell culture model of IGF-1 induced

myotube hypertrophy (Chapter 3).

2. Optimise a C2C12 cell culture model of TNF induced myotube atrophy (Chapter 4).

3. Determine to what extent TNF induced JNK signalling plays a part in mediating TNF

induced myotube atrophy and inhibiting IGF-1 induced hypertrophy (Chapter 5).

4. Test the effectiveness of the JNK inhibitor TAT-TIJIP in vivo on reducing exercised

induced myofibre necrosis (Chapter 6).

Hypothesis

The central hypothesis of the thesis is that the damaging effects of TNF on skeletal

muscles are mediated by interference, via JNK, with IGF-1 receptor signalling. It was

further proposed that:

1. Administration of TNF to myotubes in tissue culture will induce myotube atrophy

and will prevent IGF-1-mediated myotube hypertrophy.

2. Inhibition of TNF activated JNK (using JNK inhibitors) will prevent the TNF-

mediated myotube atrophy in tissue culture.

3. In vivo administration of JNK inhibitors will reduced TNF-mediated necrosis of

dystrophic muscles in mdx mice.

Chapter Two Materials and Methods

24

Chapter Two

Materials and Methods

2.1 Cell culture

In this research, immortalised and primary muscle cell cultures were used. Immortalised

cell lines were commercially purchased from the American Type Culture Collection

(ATCC; Manassas, USA). Primary muscle cell cultures were prepared from mice as

detailed below in Section 2.1.3.1. Primary cultures were included as a skeletal muscle

cell model that bears closer resemblance to muscle in vivo, compared to immortalised

cultured rodent muscle cell lines, and provided a useful additional in vitro model to

study the morphological effects of treatments on myotubes.

2.1.1 Immortalised muscle cell lines

2.1.1.1 C2C12 murine myotube cultures

The C2C12 murine skeletal myoblast (Yaffe and Saxel 1977) cell line was purchased

from the ATCC (Manassas, USA). In this study, two batches (with different passage

numbers) of C2C12 myoblast cultures were used. Initial optimisation experiments were

performed on C2C12 myoblasts that had been through at least 25 passages (actual final

passage number unknown). Due to the high passage number, these cells were not

suitable for further studies and a new batch of cells was purchased. Final hypertrophy

experiments and subsequent signalling studies were performed using these cells at

passages 4 to 6.

2.1.1.2 L6 rat myotubes cultures

The L6 rat skeletal myoblast (Yaffe 1968) cell line was provided by the Department of

Biochemistry, The University of Adelaide, Adelaide, South Australia (1992). The

passage number of these cultures is unknown.

2.1.2 Animal strains

All strains of mice were obtained from the breeding colonies established at the Animal

Resource Centre (ARC) in Murdoch, Western Australia. The mice were bred under


25

specific pathogen free conditions. The mice were housed in standard animal cages at the

Biological Sciences Animal Unit, at the University of Western Australia. The mice were

maintained at 12-hour day-night cycle and allowed access to standard chow and

drinking water ad libitum. All experiments were conducted in strict accordance with the

guidelines of the University of Western Australia Animal Ethics Committee and the

National Health and Medical Research Council, Australia.

2.1.2.1 Animals used for primary muscle cell culture preparations

For the preparation of primary muscle cell cultures, adult 4-5 weeks old transgenic mice

which over-express Class 2 IGF-1 Ea (IGF:C2) in skeletal muscles (Shavlakadze et al.

2005) and the background FVB strain (a wild-type control) were used.

2.1.2.2 Animals used to test the effects of the JNK inhibitory peptide TAT-TIJIP

Adult 6 week old male dystrophic mdx mice (c57BL/10ScSnmdx/mdx) were used to test

the protective effects of the JNK inhibitor TAT-TIJIP in vivo (see Chapter 6).

2.1.3 General cell culture conditions

2.1.3.1 Preparation of primary cell cultures

Myoblasts were isolated from skeletal muscles of 4 to 5 week old IGF:C2 and FVB

mice to establish the primary muscle cultures. The mice were sacrificed and dipped in

70% (v/v) ethanol. The muscles from the legs, hips and back were then excised and

placed in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, Vic, Australia)

containing 20 µg/ml gentamycin antibiotic (Sigma-Aldrich, NSW, Australia). The

muscles were then rinsed with 3-4 changes of Phosphate Buffered Saline (PBS; 145mM

NaCl, 7.50mM Na2HPO4, 2.85mM NaH2PO4.2H20 pH 7.4) before being placed in a

sterile petri dish where special care was taken to remove tendons and nerves from the

muscle.

The muscles were then transferred to a petri dish and minced with scissors in

dissociation buffer (200U/ml collagenase type 1, and 200 U/ml dispase in 200ml

DMEM) at room temperature (RT) for 5 min. The dissociation buffer with released cells

was collected and mixed with a further 20 ml of dissociation buffer and placed in a flask

for further steps in trypsination. The tissue was digested by placing the flask on a pre-

warmed stirring plate in a 37ºC incubator for 45 min with stirring. The solution was


26

transferred to a 50 ml tube and centrifuged at 1500 rpm for 5 min at RT. The

supernatant was discarded and the pellet washed twice by re-suspending in PBS and re-

centrifuging at 1500 rpm, for 5 min at room temperature. The cell pellet was re-

suspended in 20 ml Trypsin/EDTA (Sigma-Aldrich), transferred to a clean flask and

placed at 37ºC with stirring for 10 min. Trypsin was inhibited by the addition of 1ml

FCS. The solution was transferred to a 50 ml tube and centrifuged for 5 min at 1500

rpm at RT. The cell pellet was resuspended in 20 ml primary cell growth medium

consisting of DMEM supplemented with 20% foetal calf serum (FCS, Sigma-Aldrich)

1% GlutaMAX-1 (Invitrogen), 1% penicillin/streptomycin Invitrogen) and 12.5 ng/ml

basic Fibroblast Growth Factor (bFGF; Sigma-Aldrich), and filtered through a 0.2 µm

sterile filter. The solution was then transferred to a 0.1% collagen coated flask and

incubated for 2 h at 37ºC in a saturated humidity atmosphere containing 5% CO2/95%

air in an incubator (Crown Scientific, WA, Australia) (This is pre-plate 1; PP1).

To remove contaminating fibroblasts, the supernatant with un-adhered cells was

transferred to another collagen coated flask with medium replenished (10 ml of primary

cell proliferation medium) at 37ºC, 5% CO2/95% air atmosphere for a further 24 h

(PP2). This process of pre-plating was continued for a further 3 days. By PP5-6,

myoblast purity was verified as > 80% through immunohistochemical staining with

monoclonal mouse anti-desmin clone DE-U-10 (D1033, Sigma-Aldrich) as described in

Section 2.4.2.

2.1.3.2 Resurrection of cyropreserved cell stocks

Immortalised skeletal muscle (C2C12 and L6) myoblasts were resurrected from

cryopreserved stocks, which had been frozen down at concentrations of approximately 1

x 105 cells/ml in 1 ml of proliferation (high serum) medium, consisting of DMEM

supplemented with 20% FCS and 1% penicillin/streptomycin, with the addition of 10%

dimethylsulphoxide (DMSO; Sigma-Aldrich). The cell stocks were stored in liquid

nitrogen until required.

Cells were thawed on ice and resuspended in 9 ml of proliferation medium under sterile

conditions in a laminar flow biological cabinet. The medium was pre-warmed in a 37ºC

water bath prior to use. Cells were then centrifuged at 1500 rpm for 3 min using an

Eppindorf Centrifuge 5702 (Crown Scientific); the supernatant was then discarded and


27

the cells resuspended in 10 ml of proliferation medium. The cell suspension was then

transferred to a 75cm2 flask. For proliferation, cells were grown in 10 ml proliferation

medium at 37°C in 5% CO2/95% air atmosphere.

2.1.3.3 Trypsinising and seeding cells onto plates

Myoblasts were cultured until they reached 70% confluence; the proliferation medium

was then removed and the cells were washed once with 4 ml of sterile PBS. This

ensured that all remaining media was removed, as this can interfere with the

Trypsin/EDTA reaction. PBS was discarded and 4 ml of Trypsin/EDTA solution was

added to the flask. This was gently agitated by hand for 2 to 3 min causing cell rounding

and detachment from the surface of the flask, cell detachment was confirmed by

microscopic examination. The Trypsin reaction was stopped by adding 6 ml of

proliferation medium; the cell suspension was then pelleted at 1500 rpm for 3 min in a

sterile 15 ml Falcon tube in an Eppindorf Centrifuge (model 5702). The supernatant was

then discarded and the cells resuspended in a 1 ml proliferation medium. Ten microliters

of cell suspension was removed and mixed with 90 µl of Trypan Blue solution (Sigma-

Aldrich), resulting in a 1 in 10 dilution. The extent of dilution in Trypan Blue was

dependant on the size of the pellet observed after centrifugation. Ten microliters of the

Trypan Blue/cell solution was placed on a Neubauer haemocytometer to determine the

total cell count. Cells that had a blue appearance were considered dead and not counted

(Figure 2.1). The four individual squares were counted separately and the total count

was averaged. The concentration of the cell suspension is calculated according to

Formula 1:

Formula 1: Cells/ml = sum of cells in four squares x dilution factor x 104

4

The multiplication factor (104) is derived from the volume of each of the four counted

squares, which are 0.1 mm deep and 1 x 1 mm square. This equates to a volume of 0.1

mm3 or 0.0001 ml (10-4 ml).


28

Figure 2.1: Neubauer haemocytometer. Cells are counted in the four large squares (A,B,C and D) which are further subdivided into 16 smaller squares. The average number of cells is taken and multiplied by 1 x 104 and the Trypan blue dilution factor. Cells are only counted on the lower and right hand lines (5 grey cells), with those falling outside, on top of left line (3 whites cells) omitted.

The amount of cell suspension required depended on the intended use of the cells and

the size and number of wells to be seeded, and was diluted to the appropriate volume in

proliferation medium (Table 2.1). Generally (except during the optimisation of plating

conditions, see Section 3.1.1) for the morphology studies, C2C12 myoblasts were plated

at a concentration of 1 x 105 cells per dish in proliferation medium, and primary muscle

cells were plated at 2.5 x 105 cells per dish in primary cell growth medium. Cells were

plated on glass cover slips (Menzel-Glaser, Germany) and coated with a 0.01% collagen

(type I) solution (Invitrogen) (diluted in PBS) in 35 mm2 plastic dishes (Falcon). In the

initial tissue culture experiments (detailed in Section 3.1.1), dishes were also coated

with 0.1 mg/ml Poly-D-Lysine (PDL) (Chemicon, Vic, Australia) diluted in PBS. For

studies requiring protein extraction (Cell signalling studies: Chapter 5), C2C12

myoblasts were plated at a concentration of 5 x 105 cells/dish in 60 mm2 uncoated

plastic dishes (Table 2.1).


29

Table 2.1 Dimensions of various tissue culture plates and the myoblast plating densities used in

these studies.

Usage of cells Size of well/dish Growth area (cm2)

Growth volume

Cell density (cells/well)

C2C12: 1 x 105 cells Morphology Studies

35 mm2 dish 8 2 ml Primary: 2.5 x 105 cells

Protein Extraction

60 mm2 dish 21 5 ml 5 x 105 cells

Cell fusion was induced by changing the medium from proliferation to low serum

medium (DMEM supplemented with 2% horse serum (HS)) at 48 h after plating, or

once cells had reached 70% confluence; this is termed ‘day 0’ of fusion. Fusion medium

was changed every 48 h until treatment commenced.

2.2 Reagents and Antibodies

2.2.1 Treatment reagents

Analogues of IGF-1; ‘LONGTMR3IGF-I’ and Des(1-3)IGF-1 were purchased from

GroPep (SA, Australia). Mouse recombinant Tumour necrosis factor-alpha (TNF) was

purchased from Chemicon (Vic, Australia) and both Anisomycin (A 9789), a control for

stimulating phosphorylation of c-Jun N terminal Kinase (JNK) (Cuenda and Cohen

1999) (described in detail in Section 5.1) and Insulin (15500) were purchased from

Sigma-Aldrich.

2.2.2 JNK inhibitory peptides

The ATP competitive inhibitor of JNK SP600125 was purchased from Sigma-Aldrich

and AS601245 was purchased from Sapphire Bioscience (NSW, Australia). The TAT

control peptide; a cell permeable 10 amino acid Tat cell transport sequence (TAT) and

the cell permeable JNK inhibitory peptide TAT-TIJIP; a shorter peptide inhibitor

comprising of an 11 amino acid sequence (Truncated Inhibitor region of JNK

Interacting Protein; TIJIP) linked to TAT, were all synthesised by AusPep (Parkville,

Australia). Peptide purity was at least 80% as determined by mass spectrometry. The

sequence of TAT-TIJIP was GRKKRRQRRRPPRPKRPTTLNLF, with the sequence

of TIJIP indicated in bold and underlined.


30

2.3 Antibodies

2.3.1 Western blotting

Primary antibodies against phospho-JNK (#4671) and total JNK protein (#9258) were

purchased from Cell Signaling Technologies (Beverly, MA, USA). Phospho-AKT (Ser

473, #4058S) and AKT (#9272) were purchased from Cell Signaling Technologies,

IRS-1 (IRS-1 c20:sc-559, Santa Cruz, CA, USA ). Monoclonal phosphotyrosine pY99

(sc-7020, Santa Cruz) and the secondary horseradish peroxidase (HRP)-conjugated anti-

rabbit antibody were purchased from Sigma-Aldrich (NSW, Australia).

2.3.2 Immunohistochemistry

Primary antibodies were obtained as follows: monoclonal mouse anti-desmin clone DE-

U-10 (D1033, Sigma-Aldrich) and mouse polyclonal phospho-c-Jun (#9165, Ser 63/73)

antibody (Cell Signaling). Secondary antibodies were goat anti-mouse IgG conjugated

to Alexa Fluor 488 (A11001, Molecular Probes, Invitrogen, Australia) and donkey anti-

rabbit IgG conjugated to Alexa Fluor 594 (A21207, Molecular Probes).

2.4. Myotube morphology studies

2.4.1 Treatment regime for myotube cultures

Due to the slow rate at which the myoblasts fused into myotubes, C2C12 cells were

treated on day 7 of fusion, whereas treatment of the primary myotube cultures began on

day 4 after fusion medium (see Chapter 4). An analogue of IGF-1 known as

‘LONGTMR3IGF-I’ (Grow-Pep, SA, Australia) was added to the myotubes at a

concentration of 10 ng/ml, and mouse recombinant TNF (Sigma-Aldrich) was added a

concentration of 20 ng/ml. For all muscle cell cultures, the medium and treatment were

replaced at 24 h intervals for 3 days.

For studies where TNF treatment was combined with the JNK inhibitors (and respective

controls), myotubes were pre-treated for 1 h with the test JNK inhibitor before TNF

treatment was administered. Both treatments were then replaced daily for 3 days.


31

2.4.2 Immunohistochemistry

After 3 days of treatment (C2C12 - fusion day 10; primary cultures - fusion day 7) cells

(fused on glass coverslips) were fixed for 15 min in 3% formaldehyde made up in PBS.

After 3 washes with PBS, the cells were incubated for 60min at RT in blocking buffer to

minimise non-specific antibody binding: 10% FCS diluted in PBS wash buffer (0.1%

Saponin (Sigma-Aldrich); 0.5% Bovine Serum Albumin (BSA; Sigma-Aldrich)). For

the myotube morphology studies (Chapters 3 and 4) the cells were incubated with 1:300

dilution primary monoclonal mouse anti-desmin (Sigma-Aldrich) at 4ºC overnight.

Following this, the primary antibody was then removed by washing the cells 3 times

with PBS wash buffer before the addition of the appropriate secondary antibody (used at

a dilution of 1:250). Goat anti-mouse IgG conjugated to Alexa Fluor 488 (Molecular

Probes) was applied to the cells for 60 min at RT. Cells were again washed 3 times and

the nuclei were stained for 1 min with 50 µg/ml Hoechst Nuclear Stain HO33342

(Sigma-Aldrich). The cells (on the glass coverslips) were mounted on superfrost glass

slides (Menzel-Glaser) using Polyvinyl Alcohol mounting medium (PVA; P8136-7,

Sigma-Aldrich) and stored at 4ºC. The primary antibody was omitted from one of the

coverslips to serve as a negative control.

2.4.3 Image analysis: measuring myotube widths

The slides (myotubes) were viewed under a Nikon Eclipse 90i microscope with the

filters set to 485 nm excitation and 520 nm emission for green fluorescence (to visualise

desmin staining); 544 nm excitation and 590 nm emission for red fluorescence (to

visualise phospho c-Jun stain); and 357 nm excitation and 540 nm emission for blue

fluorescence (to visualise nuclear stain). Images were captured with a Photometrics

Cool Snap ES camera and personal computer with Digital Optics V++ v.4.0 software.

For each slide (representing 1 myotube dish) the total number of fields per coverslip

was calculated using the Image Pro Plus v.4.5.1.29 software (Image Pro Plus) and

assigned a number. From each coverlslip, random fields were selected for analysis using

the random number generator function in Microsoft Office Excel 2003. From each

myotube dish a total of 10 random images were captured with a 10x objective (Figure

2.2).


32

To quantify myotube widths, 5-6 myotubes were selected from each of the 10 images,

resulting in a total of 50 myotubes being counted in each dish. Before each measuring

session, the system was geometrically calibrated with an image containing an object

micrometer. For each myotube, widths were sampled 50 µm apart using measuring tools

in Image Pro Plus; an example of 1 of the 10 images that were taken from each dish is

shown in Figure 2.2. Width measurements were imported into a Microsoft Office Excel

spreadsheet and averaged for each myotube (Figure 2.2). The widths that were

measured (each spaced 50 µm apart) along the entire length of the first myotube, and the

average width for that myotube is highlighted in the red box in Figure 2.2. The average

widths for each of the 6 myotubes analysed (from this particular field) is shown in red

font in Figure 2.2. Once all the myotubes from each of the 10 images/dish had been

measured, the average widths of each myotube (in red font) were summed and then

divided by the number of myotubes counted for that dish. This then resulted in 1 mean

width calculated per dish for each dish. For each treatment, a minimum of 3 myotube

culture dishes were sampled and analysed.


33

Figure 2.2: Measuring myotube widths using Image Pro Plus software. Representative immunofluorescent photomicrograph of day 10 C2C12 myotubes stained with anti-desmin antibodies (in green) viewed using Image Pro Plus software. Routinely, 10 random images were photographed from each treatment dish and myotube widths were recorded using the software. Each width measurement was uploaded into a Microsoft Excel spreadsheet from which statistical analyses was performed. Magnification: 10x. Scale bar: 100 µm.

2.4.4 Statistical analyses

Myotube widths are expressed as means with standard error (SE +). A one-way analysis

of variance (ANOVA) and unpaired Student’s t-tests were used to assess the level of

statistical significance of the difference between treatment and control groups for tissue

culture experiments. A ‘P’ value of < 0.05 was considered significant. All statistical

analyses were calculated using Microsoft Office Excel.

2.5 Western blot quantitation of JNK phosphorylation in day 7 C2C12 myotubes

2.5.1 Treatment regime for western blot analysis of phospho-JNK

Day 7 C2C12 myotubes were serum starved for 5 h before being treated with IGF-1 (10

ng/ml), TNF (20 ng/ml) or Anisomycin (5 nM), with protein extracted at various time

points following stimulation, except for Anisomycin treatment, where protein was


34

routinely extracted 15 min following stimulation to serve as positive control for JNK

phosphorylation.

2.5.2 Extraction and sample preparation

Following treatment, C2C12 myotubes were washed once with ice cold PBS and lysed

in an ice cold stress lysis buffer (20 mM HEPES pH 7.7, 2.5 mM MgCl2, 0.1 mM

EDTA, 20 mM b-glycerophosphate, 100 mM NaCl, 0.1% Triton X 100, 500 µM DTT,

100 µM Na3VO4, 100 µg/ml PMSF, 0.01% NP40, protease inhibitor cocktail tablet

(Roche 04693159001) and incubated on ice for 20 min. Cell lysates were centrifuged at

12,000 x g and the supernatant was stored in 100 µl aliquots at -80°C until used for

analyses. Protein was quantitated using the Bradford assay (see below).

2.5.3 Protein quantitation

Protein quantitation was performed using a Bio-Rad Protein Assay Kit (Bio-Rad

Laboratories, Gladesville, NSW). The kit is based on the Bradford method, using BSA

(Sigma-Aldrich) as a standard. Protein samples were quantitated with reference to a

standard absorbance curve, which was generated by measuring the OD of known

Bovine Serum Albumin (BSA) standards. The BSA standards were prepared by serial

dilutions of BSA in PBS (Table 2.2).

Table 2.2: Concentration of BSA in the protein assay standards

Standard BSA concentration

Standard 1 100 µg/ml





Each standard was aliquoted (10 µl) in duplicate glass tubes. The samples to be

analysed were then diluted 1:10 in PBS and 10 µl of each sample was aliquoted into

new tubes in duplicates. Following this, 200 µl of Bio Rad Reagent was added to each

tube, vortexed and left to stand for 10 min at RT before being analysed using a


35

spectrophotometer (Beckman DU Series 640, Beckman Instruments Inc, Fullerton, CA,

USA). The spectrophotometer was zeroed using PBS and the samples were measured in

the visible spectrum at 750 nm. The protein content of each homogenate was calculated

from the standard curve.

2.5.4 Protein sample preparation

2.5.4.1 Whole cell lysates

Each 100µl aliquot of homogenate was mixed with 50 µl of 3 x Protein loading buffer

(1 M Tris pH 6.8, 6% SDS, 30% Glycerol, 0.02% β-mercaptoethanol, 6% bromophenol

blue), briefly vortexed, heated at 95ºC for 5 min, and then chilled on ice until loaded

onto the gel.

To calculate the volume of the sample to be loaded, a simple formula was used. For

examples: to load 100 µg of protein from a homogenate sample (concentration of 6

µg/µl), into a well in the gel that holds a maximum of ~40 µl, 25 µl of the sample (in

protein loading buffer) was combined with 10 µl of PBS, bringing the total volume of

the sample to 35 µl. To avoid spillage all samples were made up to ~35 µl using PBS.

2.5.4.2 Immunoprecipitation of IRS-1

For the immunoprecipitation (I.P.) of IRS-1, following the appropriate treatment, the

cell lysates were prepared as previously described (see Section 2.5.2). Once protein

quantitation had been performed (see Section 2.5.3), immunoprecipitation was

performed from approximately 1mg (1000µg) of protein. Supernatants were incubated

with 2-5 µg of anti-IRS-1 antibody and protein A-Sepharose 4B Fast Flow beads (P-

9424, Sigma-Aldrich) for anywhere between 2-12 h (when attempting to optimise the

protocol) at 4ºC and with/without rotation and for 2 h (when performed in Dr John

Whitehead’s laboratory - Chapter 5) at 4ºC without rotation. Immunocomplexes were

then washed at least 5 times in an ice cold stress lysis buffer and resuspended in a

protein loading buffer. The samples were then subjected to SDS-PAGE.

2.5.5 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Protein samples were resolved by SDS-PAGE under denaturing conditions on 12%

(resolving) polyacrylamide gel. The preparation and composition of this gel is described

below (Table 2.3a). After ensuring that no air bubbles formed and that the gel surface


36

had set flat, the resolving gel was carefully poured in the glass plate assembly and then

overlaid with ddH2O. The gel was then left to polymerize for approximately 20 min.

Following this, ddH2O was removed and the stacking gel (composition outlined in

Table 2.3b) was poured on top of the set resolving gel.

Table 2.3a: Composition of Resolving gel: 12% polyacrylamide in 1 M Tris-HCL pH 8.8

1M Tris (pH 8.8) 3.75 ml

30% Acrylamide

(37.5:1. Acrylamide/bis) 4 ml

ddH20 2.2 ml

20% SDS 50 µl

TEMED 10 µl

APS (@ 0.2 mg/µl) 75 µl

Table 2.3b: Composition of Stacking gel: 6% polyacrylamide in 0.5 M Tris-HCL pH 6.8

1M Tris (ph 6.8) 1.25 ml

30% Acrylamide

(37.5:1. Acrylamide/bis) 2 ml

ddH20 6.7 ml

20% SDS 50 µl

TEMED 10µl

APS (@ 0.2 mg/µl) 75 µl

Spacers were inserted and the gel was allowed to set. Once the stacking gel had

polymerized, the gel casting chamber was transferred to a chamber filled with 1x

Electrode Buffer (5x Electrode Buffer, pH 8.3: 15 g Trizma Base, 72 g Glycine, 5 g

SDS made up to 1 L with ddH2O). The spacers were removed and the wells were

flushed with electrode buffer to ensure that there was no unpolymerised polyacrylamide

remaining. The samples and 15 µl of the SeeBlue Plus2 Pre-stained protein standard


37

(Invitrogen) were loaded onto the gel, the gel was then electrophoresed for 90 min at

140 V.

2.5.6 Protein transfer for western blotting

Polyvinylidene difluoride (PVDF) membrane (Amersham) was soaked in methanol and

washed with ddH2O. The membrane, filter paper and sponge pads were pre-soaked in

cold transfer buffer (3.03 g Trizma Base, 14.4 g Glycine, 200 ml Methanol made up to 1

L with ddH2O) for 30 min at 4ºC prior to transfer.

Following electrophoresis, the gel was removed from the casting chamber. The transfer

cassette was then assembled as illustrated in Figure 2.3. The gel was then placed in the

electroblotting tank and the tank was then filled with cold transfer buffer (4ºC). The

proteins were transferred from the gel to the PVDF membrane via electrophoresis for 90

min at 100 V. A frozen cooling unit was placed into the transfer chamber immediately

prior to transfer and replaced every 45 min. Following transfer, the transfer cassette

were removed from the tank and the PVDF membrane was washed in Tris Buffered

Saline (TBS pH 7.5: 12.1 g Trizma Base, 9.0 g NaCl made up to 1 L with ddH2O and

pH with 0.5 M HCl ) with 0.1% Tween-20 (TBS-T) for 15 min.

Figure 2.3: Illustration of the Western blotting transfer cassette. The cassette is inserted into the transfer cell with the gel nearest the cathode (+).

2.5.7 Blocking non-specific antibody binding

Prior to immunoblotting the membranes were incubated with blocking solution

consisting of TBS-T supplemented with 5% non-fat milk powder for 1 h at RT with

gentle agitation. The membrane was then rinsed in TBS-T for 5 min.

Sponge pad Pre-wetted filter paper Nitrocellulose membrane PAGE gel Pre-wetted filter paper Sponge pad -

+


38

2.5.8 Protein detection

The membrane was incubated with diluted primary antibody (rabbit anti phospho-JNK

#4671 or rabbit anti-JNK #9258 - Cell Signaling Technology) overnight at 4ºC. All

procedures were performed according to manufacturer protocols. Primary antibodies

were diluted 1:1000 in TBS-T with 5% BSA. Following incubation in primary antibody,

the membrane was washed 3 times for 20 min in TBS-T and incubated in secondary

antibody. The secondary antibody used for detection of the primary antibodies was goat

anti-rabbit (#31460, Pierce Biotechnology, Thermo Fisher Scientific, Sydney, Australia)

at a dilution of 1:10,000 in 5% milk TBS-T. Following incubation with the secondary

antibody, the membrane was washed 3 times for 20 min in TBS-T.

2.5.9 Chemiluminescent signal detection to observe protein

The protein signal was detected by chemiluminescence using ECL detection kit (Pierce

Biotechnology). Membranes were incubated for 5 min in the SUPERSIGNAL substrate

working solution (1:1 mix of lumino/enhancer solution and stable peroxiodase solution).

Following incubation, the chemiluminescent signal was detected using Kodak Image

Station 200 mm.

2.5.10 Densitometry for AKT signalling study

Gels were scanned using a Hewlett Packard HP ScanJet 5370C and the images saved as

TIFF files at 300dpi resolution. Densitometry was performed on TIFF images using the

NIH Image freeware program ‘ImageJ: Image Processing and Analysis in Java”

(http://rsb.info.nih.gov/ij/).

2.6 Animal studies to test the effects of TAT-TIJIP

Adult 6 week old male dystrophic mdx mice (c57BL/10ScSnmdx/mdx) were obtained from

the Animal Resource Centre, Murdoch, Western Australia and maintained as previously

described (see Section 2.1.2).

2.6.1 Exercise induced muscle damage in adult mdx mice

To determine the effects of the JNK inhibitor TAT-TIJIP on exercise induced

dystrophic muscle necrosis (Chapter 6), male mdx mice (6 weeks of age) were caged

individually in 32x25x15cm rat cages and given access to a standard metal mouse


39

exercise wheel for 48 h. Wheels had a circumference of 30 cm and hung from the roof

of the cage to avoid possible obstruction from bedding (Figure 2.4). The amount of

voluntary exercise was calculated as distance travelled over 2 nights with a Huffy

bicycle pedometer that detected the number of wheel rotation via a small magnet

attached to the wheel (Radley and Grounds 2006).

Figure 2.4: Exercise equipment for voluntary running. Yellow arrows indicate wheel with magnet attached and white arrows indicate bike distance recorder attached to cage. Image adapted from Waters (2006).

The mice were separated into two groups; one group was treated with 10 mg/kg body

weight TAT-TIJIP while the control group received ddH20 only (diluent for TAT-TIJIP)

via intra-peritoneal injections using a 27 G x 0.5 inch needle 24 h prior to exposure to

voluntary exercise (before being placed in the cage fitted with the wheel).

2.6.2 Animal anaesthesia and sacrifice

Mice were humanely anesthetised with a gaseous mixture of 1.5% Rodia Halothane

(Merial, Paramatta NSW, Australia) N2O and O2, and killed by cervical dislocation.

2.6.3 Tissue sampling and processing

For paraffin processing, the right leg of each mouse was fixed by immersion in 4%

(w/v) paraformaldehyde (Sigma-Aldrich) (pH 7.6) diluted in PBS, for a minimum of 24

h. Quadriceps muscles were dissected (see Section 6.1) from the fixed legs, transferred

to 70% ethanol and processed in a Shandon automatic tissue processor overnight. The

muscles were bisected in the mid-region and embedded with both cut surfaces at the top

of the paraffin block for transverse sectioning.

A B


40

Paraffin blocks were transversely cut into 5 µm thick sections using a Leica retractable

microtome. The sections were stretched on a water bath containing 0.1% HS (1 ml HS

diluted in 1000ml H2O) at approximately 42ºC and collected on silane (3-aminopropyl-

triethoxysilane)(Sigma-Aldrich) coated glass slides (Menzel-Glaser). Slides were air

dried for approximately 30 min and placed in the oven at 37 ºC overnight to ensure

proper attachment of the section to the glass slide.

2.6.4 Haematoxylin and eosin staining

Paraffin sections were dewaxed in three changes of xylene (MERCK, Biollab, Vic.,

Australia) (2 min each) and re-hydrated by passing through graded alcohol and ddH2O:

2 changes of 100% ethanol (2 min each); 1 change of 70% ethanol (2 min); and 1

change ddH2O (1 min). Re-hydrated slides were placed in Haematoxylin (BDH

Laboratory Supplies, Biollab, Vic., Australia) (30 sec) and washed in tap water.

Haematoxylin stained slides were placed in 70% ethanol (2 min), stained in Eosin

(MERCK) (20 sec), washed, and then dehydrated in 3 changes of 100% ethanol (2 min

each) and passed through 2 changes xylene (3 min each). Slides were mounted with

PVA mountant for microscopy (BDH Laboratory Supplies) and air dried.

2.6.5 Image analysis: quantification of necrosis

Non-overlapping, bright field images of transverse mdx muscle sections were captured

with a LEICA DMBRE microscope (at 10x magnification), Nikon Digital Camera

DXM1200F and personal computer using the Image-Pro Plus v.4.5.1.29 software to

view the entire muscle cross section. Areas of muscle necrosis were identified using the

Image-Pro Plus v.4.5.1.29 software by the presence of dark blue stained inflammatory

cells (basophilic staining) and/or the fragmented sarcoplasm of necrotic myofibres (Fig

2.5). The area of necrosis was recorded as a percentage of the whole section (Radley

and Grounds 2006).


41

Figure 2.5: Typical histology of dystrophic mdx quadriceps muscle. (A) Unexercised mdx muscle, characterised by regenerated myofibres of different sizes with central nuclei (arrows) with a low level of necrosis (circled). (B) Exercised mdx muscle characterised by increased areas of myofibre necrosis (indicated within area outlined by broken line) identified by the infiltration of inflammatory cells (arrow) and fragmented sarcoplasm (asterisk). Scale bar, 100µm

2.6.6 Statistical analyses

All data are expressed as means + Standard Error. A two-way analysis of variance

(ANOVA) was used to assess differences between TAT-TIJIP treated and untreated

control groups for the mdx mice exercise experiment.

*

*

A B

Chapter Three Validation of cell culture models of IGF-1 induced skeletal muscle hypertrophy

42

Chapter Three

Validation of cell culture models of IGF-1 induced skeletal

muscle hypertrophy

Introduction

The purpose of this study is to validate the morphological effects of exogenous IGF-1

on skeletal muscle myotubes in tissue culture. Initial experiments focused on

establishing a skeletal muscle cell culture model that would respond to stimulation with

IGF-1. This same model was then used in TNF induced atrophy and combined with

further IGF-1 studies (see Chapter 4). The experimental design was based on published

methods reporting that addition of an analogue of IGF-1, LONGTMR3IGF-I, in tissue

culture to differentiated cells of the immortalised mouse C2C12 skeletal muscle cell line

(Yaffe and Saxel 1977) induced myotube hypertrophy (Florini et al. 1996; Rommel et

al. 1999; Rommel et al. 2001; Sultan et al. 2006). Validation of these findings is

necessary to confirm that the C2C12 cells are a suitable model to investigate the

morphological and protein changes that result from IGF-1, TNF and combined IGF-

1/TNF treatments.

Optimisation of fusion conditions was conducted on the C2C12 skeletal muscle cells

(see Section 3.1). The effect of various doses of IGF-1 treatment was tested on two

muscle cells lines; the C2C12 adult mouse, and L6 neonatal rat cell line (Yaffe 1968).

While the IGF-1 receptor is present in both cell lines, the expression of IGF-1 varies

between them; C2C12 cells express IGF-1 (Tollefsen et al. 1989), whereas L6 cells

have little or no IGF-1 expression (Engert et al. 1996). In addition those two cell lines

show altered expression of a range of myogenic factors; L6 cells have restricted

expression of myogenic factors, whereas C2C12 cell have a more normal profile of gene

expression (Engert et al. 1996).


43

Results

3.1 Establishing optimum culture and treatment conditions

3.1.1 Comparing C2C12 myotube formation on different substrates

Initial experiments were designed to optimise culture conditions for cell attachment,

proliferation and differentiation of mouse C2C12 muscle cells when plated in 35 mm2

plastic dishes (Falcon) and thereafter on glass cover slips. Dishes were pre-coated with a

0.01% collagen (type I) solution (Invitrogen) (diluted in PBS) or with 0.1 mg/ml Poly-

D-Lysine (PDL) (Chemicon). Myoblasts were plated at a concentration of 0.5 x 105

cells per dish in a proliferation (high serum) medium, consisting of Dulbecco’s

Modified Eagle Medium (DMEM; see Section 2.1) supplemented with 20% (v/v) foetal

calf serum (FCS). For this initial experiment, fusion was induced by changing the

medium to low serum medium (DMEM supplemented with 2% horse serum (HS) at 48

h after plating. The cells used in these preliminary experiments had been through a

minimum of 25 passages, however, as previously noted, the exact passage number of

these cells was not known.

After 3 days of culture in low serum medium, the fusion of myoblasts into myotubes

was only present when the cells were cultured in dishes that had been pre-coated with

0.01% collagen (type I) (Figure 3.1: A), but not with PDL (Figure 3.1: B) or uncoated

(plastic) dishes (Figure 3.1: C). Cells plated in the PDL and uncoated dishes did not fuse

and few cells remained attached after 3 days of culture in the low serum medium.

Compared to the numbers of cells that were plated, only half the number of cells were

attached in the PDL dishes, with less than half attached in the uncoated dishes (Figure

3:1: B and C).


44

Figure 3.1: Myotube formation on different surface coatings. Phase contrast images of C2C12 cells photographed at 3 days after medium was changed from proliferation (high serum) to fusion (low serum) medium. Myoblasts had aligned and fused into myotubes in the petri dish pre coated with 0.01% collagen (type I) (A), whereas cells remained unfused in the Poly-D-Lysine coated dish (B). Most cells plated in the uncoated petri dish (C) had lifted off. Magnification: 10x. Bar: 100 µm.

3.1.2 Determining the optimum fusion medium and seeding density for C2C12

myotube formation

To determine the optimum medium for inducing the fusion of C2C12 myoblasts into

myotubes, cells were plated in DMEM supplemented with, or without HS, to a final

concentration of 2%. Under these fusion conditions it was also necessary to determine

the seeding density at which overlap of C2C12 myotubes was minimised, so that

measuring individual myotubes would be possible for later morphological analysis.

Myoblasts were plated at 2 cell densities (0.5 x 105 or 1 x 105 cell per well) and cultured

in proliferation medium (DMEM + 20% FCS) for 48 h, following this the medium was

changed to fusion medium (DMEM without or with 2% HS).

At the lower cell density, the addition of horse serum to the fusion medium resulted in

the formation of slightly more myotubes (Figure 3.2: A) compared to cultures without

horse serum (Figure 3.2: C). When myotube formation was induced with the addition of

horse serum, alignment and fusion of the myoblasts into myotubes was more uniformly

spread within the culture (Figure 3.2: A and B). Cells cultured in the DMEM only

medium formed shorter and less evenly distributed myotubes compared to cells cultured

in the presence of horse serum (Figure 3.2: C and D). Furthermore, after 3 days of

culture in the DMEM only medium, the myotubes were more susceptible to lifting off

the dish with few myotubes remaining, compared to the horse serum supplemented

cultures.


45

The differences in the formation of myotubes in medium supplemented with or without

horse serum were subtle (Figure 3.2). However, when comparing the seeding densities

at which the cells were plated, the benefits on formation and continuity of myotubes

within dishes that were fused in the presence of horse serum was evident (Figure 3.2: B

and D). The horse serum supplemented cultures produced longer myotubes, which is

more desirable for subsequent morphological analyses. In this medium (DMEM + 2%

HS - hereafter referred to as fusion medium), the seeding density of 1 x 105 cells/well

was optimal for the formation of non-overlapping myotubes, which were able to be

cultured for up to 5 days without lifting off the dishes.

Figure 3.2: Fusion of C2C12 myoblasts into myotubes in two media (DMEM +/- HS) at two different initial plating densities in 35mm collagen (type I) coated dishes. Phase contrast images show C2C12 myotubes at day 3 of culture in Dulbecco’s Modified Eagle medium (DMEM) medium supplemented with horse serum (HS) (A and B) and without (C and D). More myoblasts fused into myotubes in horse serum supplemented medium, when plated at initial cell densities of 0.5 x 105 (A) and 1 x 105 cell/well plated (B) compared to horse serum free cultures (C and D). At an initial cell plating density of 1 x 105 cell/well, myotubes in the presence of horse serum (B) were longer and more uniformly spread than those in horse serum free medium (D). Magnification: 10x. Bar: 100 µm.


46

3.2. Preliminary studies to determine the biological activity and optimum

dosage of IGF-1 required for inducing hypertrophy in C2C12 and L6 myotube

cultures (passage > 25)

After optimising the seeding density and fusion media conditions, it was necessary to

determine if the C2C12 cultures were responsive to treatment with IGF-1 and, if so,

what was the optimum dosage for this to occur. This was evaluated by comparing the

phenotypic changes in myotube size in untreated and IGF-1 treated cultures. The

analysis was initially performed on C2C12 and then on L6 myotubes. Both these cell

lines had been through a minimum of 25 passages and were defined as ‘old’ cultures.

Two analogues of IGF-1 were tested; LONGTMR3IGF-I and Des(1-3)IGF-1. All studies

were conducted in triplicate.

3.2.1 Effects of IGF-1 (LONGTMR3IGF-I) treatment on day 3 mouse C2C12

muscle myotubes (passage > 25)

To compare results from this study and validate previous data by Florini et al (1996) it

was necessary to administer the same analogue and treatment dosage of IGF-1 to

differentiated C2C12 myotubes. Florini et al (1996) treated cells with an analogue of

IGF-1 known as ‘LONGTMR3IGF-I’ that has a lower affinity for IGF Binding Proteins

(IGFBPs) compared to IGF-1 (Francis et al. 1992). This is due to the addition of a 13

amino peptide at the N-terminus and the substitution of glutamic acid (E) at position 3

with arginine (R). For the remainder of the tissue culture studies this analogue is

referred to as ‘IGF-1’.

The C2C12 myoblasts used in this part of the study routinely fused to become myotubes

after 3 days of culture in low serum media (DMEM + 2% HS) when plated at an initial

seeding density of 1 x 105 cells/well (see above). This was termed day 3 of

differentiation. Myotubes were treated on day 3 for a further 3 days with IGF-1 at a

dose of 10 ng/ml, the standard dosage according to previously published hypertrophy

studies (Florini et al. 1996; Rommel et al. 1999; Rommel et al. 2001). Treatment of the

myotubes with IGF-1 at dosages of 20 ng/ml and 40 ng/ml were also included in the

study to determine if hypertrophy could be induced at a higher dosage than that

routinely administered (Florini et al. 1996; Rommel et al. 1999; Rommel et al. 2001).

Medium and treatment were replaced daily to ensure that any decrease in stability of


47

IGF-1, when in the culture medium, did not interfere with the interpretation of the

results for the effects of IGF-1.

Typical phase contract microscopy images of day 6 treated and untreated myotubes

(Figure 3.3) showed no noticeable change in the size of the treated myotubes, when

treated with IGF-1 at a dose of 10 ng/ml, 20 ng/ml of 40 ng/ml, compared to untreated

controls. Where myotubes had been fused in DMEM with 2% horse serum (DMEM +

HS) and treated with IGF-1 at a dose 20 ng/ml (Figure 3.3: E), small formations were

apparent which resembled ‘myosacs’; this is where the cells are branched and contain

many myonuclei (Yotov and St-Arnaud 1996; Palmer et al. 2001). These formations,

however, were only observed in one of the three myotubes culture replicates treated

with 20 ng/ml IGF-1.

Even though the optimum fusion medium for inducing fusion and propagating the

growth of C2C12 myotubes was previously determined to be DMEM + HS (see above),

in this study the myotubes were also cultured and treated in horse serum free medium to

asses whether serum starvation of the myotubes might amplify the hypertrophic effects

of IGF-1. The treated myotubes cultured in the serum free medium (DMEM only) did

not show any changes in size compared to those treated and cultured in horse serum

(Figure 3.3).

3.2.2 Effects of Des(1-3)IGF-1 treatment on day 3 mouse C2C12 muscle

myotubes (passage > 25)

As hypertrophy of C2C12 myotubes was not observed after 3 days of treatment with

IGF-1 (LONGTMR3IGF-I), a second analogue of IGF-1 (Des(1-3)IGF-1) was tested.

Des(1-3)IGF-1, as discussed previously (Section 1.4.1.2), has a sequence identical to

human or bovine IGF-1 apart from the removal of the amino tripeptide (Gly-Pro-Glu)

(Ballard et al. 1987). Des(1-3)IGF-1 is similar to the LONGTMR3IGF-I analogue as it

has a high binding affinity for the IGF-1 receptor (Ballard et al. 1987) and low binding

affinity to IGFBPs (Ross et al. 1989).

Myotubes at day 3 of differentiation were treated with either 10 ng/ml or 20 ng/ml of

Des(1-3)IGF-1 for a further 3 days. All of the treated myotubes showed no change in

size or length compared to the untreated controls (Figure 3.4). Treatment with Des(1-


48

3)IGF-1 at a dose of 10 ng/ml was found to induce the branching of a small number of

myotubes, irrespective of the medium conditions (Figure 3.4: B and E, indicated by

arrows).

Figure 3.3: Effects of IGF-1 treatment on C2C12 myotubes. Phase contrast microscopy images of myotubes, differentiated in both horse serum (HS) and serum free medium, and treated for 3 days with IGF-1 at a dosage of 10 ng/ml (C and D), 20 ng/ml (E and F) or 40 ng/ml (G and H) show no observable hypertrophy compared to untreated myotubes (A and B). The formation of ‘myosacs’ was depicted in one of the cultures treated with IGF-1 at a dose of 20 ng/ml (E). Magnification: 10x. Scale bar: 100 µm.


49

Figure 3.4: Effects of Des(1-3) IGF-1 treatment on C2C12 myotubes Phase contrast microscopy images of myotubes, differentiated in DMEM supplemented with horse serum (HS) or in serum free medium, and treated for 3 days with Des(1-3) IGF-1 at a dosage of 10 ng/ml (B and E) or 20 ng/ml (C and G) show no change in myotubes size compared to untreated myotubes (A and D). Myotube branching (indicated by arrows) was displayed in cultures treated with 10 ng/ml IGF-1 (B and E). Magnification: 10x. Scale bar: 100 µm.

3.3 Effects of IGF-1 treatment on day 3 rat L6 myotubes (passage > 25)

As the C2C12 cells (passage > 25) did not respond to treatment with either analogue of

IGF-1 (Section 3.2.1 and 3.2.1) irrespective of the dosage, the experiments with IGF-1

were repeated using the rat L6 cell line. Considering that the aim of this study was to

validate the cell models of hypertrophy detailed in publications that used the IGF-1

analogue, LONGTMR3IGF-I (Florini et al. 1996; Rommel et al. 1999; Rommel et al.

2001) experiments testing Des(1-3)IGF-1 were not pursued.

In a striking contrast with the C2C12 myotubes, the L6 cells responded to treatment

with IGF-1 (LONGTMR3IGF-I); myotube size increased over the 3 days of treatment

(Figure 3.5: B, C, E and F). Treatment with 20 ng/ml IGF-1 induced myotube

hypertrophy to cells differentiated in both media (Figure 3.5 C and F), although this

hypertrophy was more pronounced in the horse serum free cultures (Figure 3.5: F).


50

IGF-1 treatment at the lower dose of 10 ng/ml induced the greatest size changes to the

myotubes, regardless of the medium conditions (Figure 3.5: B and E): Many of the

myotubes appeared to have fused into myotube sheets and displayed a flat and wide

appearance, compared to the long and thin untreated myotubes controls (Figure 3.5: A

and C) .

Figure 3.5: Effects of IGF-1 treatment on L6 myotubes. Phase contrast microscopy images of L6 myotubes treated for 3 days with IGF-1 at various dosages. Untreated controls show thin myotubes after 6 days in fusion media alone (A). The formation of extremely large myotubes was seen in the cultures treated with IGF-1 at a dose of 10 ng/ml (B and E), yet fewer hypertrophic myotubes and the appearance of unfused cells are seen in 20 ng/ml treated cultures (C and F). Magnification: 10x. Scale bar: 100 µm.

The hypertrophic myotubes resulting from treatment with IGF-1 at 10 ng/ml appeared

typical of those observed when IGF-1 was added to differentiating L6 cells (Coolican et

al. 1997). The myotubes were also similar to C2C12 myosacs previously described

(Yotov and St-Arnaud 1996; Palmer et al. 2001) in that they contained large areas of

dense nuclear clustering, as visualised by immunostaining with Hoechst nuclear stain

(Figure 3.6: indicated by arrows in B and D). In the DMEM only cultures, areas of

nuclear clustering were also shown (Figure 3.6: indicated by asterisks in C). Clustering

was not observed in the myotubes cultured in DMEM + 2% HS. The areas of nuclear

clustering present in the DMEM only cultures, however, considerably smaller than

those observed after the addition of IGF-1 (Figure 3.6: C compared to D).


51

Figure 3.6: Immunostained L6 myotubes treated with IGF-1. Myotubes were immunostained with anti-desmin antibody (green) and counter stained with Hoechst nuclear stain (blue). Untreated controls (A and C) remain as long thin myotubes whereas in IGF-1 treated cultures (B and D) myotubes are bigger and contain large areas of mass nuclear clustering (arrows). Magnification: 20x. Scale: 100 µm.

For the L6 cells a dose of 10 ng/ml IGF-1 appeared optimal for inducing myotube

hypertrophy over 3 days; this finding accords with literature on C2C12 myotubes

(Florini et al. 1996).


52

Discussion

Although the morphological effects of IGF-1 on skeletal muscle in C2C12 cell cultures

has been extensively researched (and to a lesser extent in L6 cells) it was necessary to

validate these findings and determine which of these cell lines was most suitable for the

proposed research, which was to develop a skeletal muscle cell culture model that could

be used to morphologically assess and quantify the effects of exogenous IGF-1

treatment and later TNF treatment (Chapter 4). The first aim of this study was to

optimise a skeletal muscle cell culture model of hypertrophy so that increases to

myotube size could be easily measured and compared with to control cultures.

The initial experiments concentrated on optimising the plating and culture conditions

that would assist myoblast attachment and myotube formation for the C2C12 cell line

and a mouse myoblast subclone of the C2 cell line (Yaffe and Saxel 1977). This cell

line is a pure myoblast population and a well established model for skeletal muscle

differentiation (Morgan et al. 1992; Lloyd et al. 2004). It was necessary that the pre-

plating solution assisted cell attachment, but did not contain enough growth factors to

stimulate cell proliferation and differentiation, as this could interfere with results where

exogenous IGF-1 (and later TNF) is administered to the cells. On this basis, two cell

surface coatings for the plastic petri dishes, collagen (type I) and the synthetic Poly-D-

Lysine, were evaluated.

The presence of the basement membrane protein collagen (type I) allowed the formation

of C2C12 myotubes. Collagen (type I) is a heterotrimer and is commonly used to

promote cell attachment in tissue culture. On plastic surfaces, collagen (type I) has been

reported to assist differentiation of C2C12 (Lawson and Purslow 2000) and avian

skeletal myotubes (Vandenburgh et al. 1991), in addition to promoting cell adhesion for

a number of other muscle cells (Gelman and Prives 1996; Wang et al. 1996).

PDL, a positively charged molecule, is also used to enhance cell attachment to plastic

and glass surfaces. PDL has been used to promote adherence of human stem cell

derived neurons (Hayman et al. 2005) and to assist in the culture of transfected mouse

3T3 fibroblasts (Nitsch et al. 1996). However, results from this, and other studies


53

(Lawson and Purslow 2000) suggest that collagen (type I), and not PDL, is a more

suitable surface coating for culturing differentiated C2C12 cells in plastic petri dishes.

The differentiation of C2C12 and L6 cells in tissue culture requires a switch in medium

from a high serum (containing 10%-20% FCS) to a low serum containing medium (2%

HS) once cells have reached 75-80% confluence. The differentiation medium for

culturing C2C12 and L6 myotubes to be treated with IGF-1 needed to supply cultures

with the minimum amount of nutrients, so that differentiation and myotube attachment

(for a period of up to 10 days) was viable. As the components present in the serum are

not well characterized, and the presence of growth factors that may interfere with later

IGF-1 treatment are not known, the myotubes were differentiated and treated in both

horse serum supplemented, or serum free, medium. This allowed IGF-1 induced

hypertrophy in the myotubes to be compared between the two media conditions and

thus the determination of the extent of serum derived growth factors contribution to

changes to myotubes size.

The differentiation of C2C12 and L6 cells was induced in both horse serum and serum

free medium. The C2C12 cells routinely formed longer myotubes in horse serum

supplemented medium (Figures 3.2, 3.3 and 3.4), whereas in the L6 cell line the

myotube lengths were unaffected by the medium conditions (Figures 3.5 and 3.6). The

C2C12 myotubes routinely appeared larger than L6 myotubes, an observation also

reported in previous research (Lawson and Purslow 2000).

Lawson and Purslow colleagues (2000) published data that demonstrated that L6 cells

were unable to form myotubes when cultured in serum free medium; the fusion of L6

myotubes in horse serum free DMEM, however was observed in the present study

(Figures 3.5 and 3.6). The results from this and Lawson and Purslow (2000) however

both confirm that the optimal medium for inducing fusion, and sustaining myotube

attachment for L6 and C2C12 cells, is DMEM supplemented with 2% horse serum.

When cultured in the presence of horse serum, and on collagen (type I) coated dishes,

L6 and C2C12 cells have been reported to show very little behavioural variation based

on similar levels of creatine phosphokinase (CPK) (a measure of myoblast fusion)

activity (Lawson and Purslow 2000).


54

The C2C12 and L6 cell lines are routinely used for studying the growth and

differentiation of muscle because of their ability to differentiate and form myotubes

relatively quickly (within 2-3 days of culture) in low serum media. IGF-1 has widely

been reported to stimulate myotube hypertrophy in day 2-3 differentiated C2C12

myotubes at a dose of 10 ng/ml for 24 h or 48 h (Rommel et al. 1999; Rommel et al.

2001); at a dose of 100 ng/ml for 48 h (Latres et al. 2005); and at 50 nM for 72 h

(Dehoux et al. 2007). Similarly, studies using the L6E9 (a subclone of the L6 cell line)

show IGF-1 induced hypertrophy in myotubes via the transfection of a muscle specific

IGF-1 vector that is only activated during cell differentiation (Musaro and Rosenthal

1999; Musaro et al. 1999; Fanzani et al. 2006). Furthermore, the same signalling

pathways that mediate myotube hypertrophy in C2C12 cells were upregulated when

IGF-1 was administered to L6 myotubes (Li et al. 2005).

The effect of cell passage number for C2C12 cells became an area of interest in this

study when neither IGF-1 nor Des(1-3)IGF-1 induced myotube hypertrophy in C2C12

cells, whereas in the L6 cell line, treatment with IGF-1 increased myotube sizes. Hughes

et al (2007) recently discussed the critical issue of misleading results that are associated

when using high passage cultures. This appears to be the first review that highlights the

importance of passage number for studies utilising cell lines. In addition, reports

investigating the effect of both IGF-1 and TNF treatment on C2C12 myotubes

(Fernandez-Celemin et al. 2002; Dehoux et al. 2007) state in the methods that the cells

were passaged (aka split or sub-cultivated) up to a maximum of 7 times in order to

preserve cell characteristics. A review of the culturing protocols for the maintenance of

C2C12 cultures outlined by the Coriell Cell Repositories (USA; http://www.atcc.org)

raised concerns regarding the history of the C2C12 cultures used in this study. The

protocol outlined by this company makes special mention of the importance to avoid

cells becoming confluent, as this decreases the myoblastic population of the cultures.

From prior laboratory cell culture records, over many years the number of times the

C2C12 cells had been passaged and frozen down was not clearly detailed.

Due to the C2C12 cells not responding to treatment with IGF-1, whereas numerous

studies had reported IGF-1 induced hypertrophy in this cell line (Florini et al. 1996;

Rommel et al. 1999; Rommel et al. 2001; Latres et al. 2005; Dehoux et al. 2007), it was

concluded that the C2C12 cells in our laboratory may have been compromised due to


55

their high passage number. Therefore, a new stock of this specific cell line was

purchased from ATCC. Experiments with the L6 cells were not continued, for multiple

reasons, important among them was the observation that the administration of IGF-1 in

this cell line induced the formation of myosacs, rather than the preferred increase in

sizes of individual myotubes. Furthermore, the primary aim of this study was to validate

and replicate IGF-1 hypertrophy results that had been published using mouse C2C12

myotubes. The later point also strongly contributed to the decision to cease experiments

in the L6 line, with subsequent experiments instead concentrating on examining the

C2C12 line.

.

Chapter Four Morphological effects of IGF-1 and TNF treatment on cultured myotubes

56

Chapter Four

Morphological effects of IGF-1 and TNF treatment on

cultured myotubes

Introduction

New C2C12 cells, freshly obtained from ATCC, were used to further test the effects of

IGF-1 on myotube hypertrophy. These tests were conducted because of the lack of

response to IGF-1 of the late passage (>25 passages) C2C12 cells (Chapter 3). These

low (3-6) passage C2C12 cells were cultured for 7 days in fusion medium before the

treatment period, in contrast to only 3 days for the preliminary experiments in Chapter

3. This slight change in protocol was to reduce the complication of unfused myoblasts

in the cultures (more pronounced after only 3 days in fusion medium) and to ensure

more complete differentiation of the myotubes; this modification follows the report of

Tolosa et al. (2005).

After 7 days of culture in the optimised fusion medium, DMEM supplemented with 2%

horse serum (Section 3.1.2), IGF-1 was administered at a dose of 10 ng/ml for 3 days.

The myotubes were then analysed at day 10. This dose has been previously reported to

induce myotube hypertrophy (characterised by an increase in myotube diameter) within

2 days when added to day 2 differentiated C2C12 cells (Rommel et al. 2001; Latres et

al. 2005) and within 1 day when administered to day 3 C2C12 myotubes (Rommel et al.

1999).

After confirming that the C2C12 muscle cells served as a suitable model to detect and

quantify changes in myotubes size, the role of the pro-inflammatory cytokine tumour

necrosis factor (TNF) on inducing myotube atrophy (muscle wasting) was investigated.

TNF was administered at a dose of 20 ng/ml for 3 days, to day 7 myotubes, to determine

the morphological effect on myotube size.


57

Finally, the combined effects of IGF-1 and TNF were tested in C2C12 myotubes to

determine if IGF-1: (i) induced hypertrophy; (ii) could prevent or reverse TNF induced

atrophy, (ii) or conversely, if TNF could prevent IGF-1 induced hypertrophy in this in

vitro skeletal muscle system. These hypotheses were further tested using primary

cultures of skeletal muscle cells isolated from transgenic mice which over-express Class

2 IGF-1 Ea (IGF:C2) in skeletal muscles (Shavlakadze et al. 2008 - unpublished data).

Primary cultures were included as a skeletal muscle cell model that more closely

resembles muscle in vivo, compared to cell lines, and provided a useful additional in

vitro model to study the morphological effects of combined IGF-1/TNF treatment.


58

Results

4.1 Effects of IGF-1 treatment on day 7 mouse C2C12 myotubes

The new, low passage C2C12 cells clearly responded to treatment with IGF-1. IGF-1

administered to 7 day myotubes, at a dose of 10 ng/ml for 3 days, induced hypertrophy

in the new C2C12 myotube cultures. Immunofluorescence using desmin antibodies

showed wider myotubes in the IGF-1 treated cultures compared to the untreated control

cultures (Figure 4.2: B and A respectively). A detailed analysis of myofibre widths (see

Chapter 2, Section 2.4.3) confirmed that treatment with IGF-1 induced a significant

increase (P < 0.001) in myotube size (increase of approximately 25%) when compared

to untreated controls (see below; Figure 4.3).

4.2 The effect of TNF treatment on day 7 mouse C2C12 myotubes

4.2.1 Independent C2C12 myoblast fusion assay to test the biological activity of

TNF

Before testing TNF on the myotubes, an independent assay was carried out to confirm

the biological activity of TNF, based on observations that TNF inhibits myoblast

differentiation (Szalay et al. 1997). C2C12 myoblasts (passage < 5) were cultured in

growth medium for 24 h and then changed to a fusion (low serum) medium to induce

differentiation and fusion. When changing the medium, 20 ng/ml of TNF was added for

4 days and myotube formation was compared to the untreated control cultures. After 4

days it was evident that myotubes had formed in the untreated control cultures (Figure

4.1: A), whereas the addition of TNF to the myoblasts was found to inhibit myotube

formation (Figure 4.1: B).


59

Figure 4.1: Fusion assay to test biological activity of TNF. Phase contrast images of myoblasts that were cultured in low serum media (A) or in the presence of TNF (20 ng/ml) (B) for 4 days. Myotubes formed in the untreated cultures (A) but not in the presence of TNF (B). Magnification: 10x. Bar: 100 µm

4.2.2 Effects of TNF treatment on day 7 mouse C2C12 myotubes

Upon verification that the TNF was biologically active, the treatment routine previously

applied in the IGF-1 hypertrophy experiments was repeated for the TNF atrophy studies

(see above Section 4.2.1). TNF, at a dose of 20 ng/ml, was administered to day 7 C2C12

myotubes for 3 days; the medium and treatment was changed daily. After 3 days of

treatment the morphological changes to the myotubes was assessed visually using

myotubes immunostained for desmin; size was quantified morphologically by the

analysis of myotube widths and the results recorded as mean myotube widths with the

standard error (SE). The significance of the difference between the untreated and treated

samples was quantified using a series of t-tests.

Immunofluorescent microscopy images (Figure 4.2) show that TNF treatment not only

reduced myotube width, but also myotube numbers. Myotubes cultures treated with

IGF-1 (Figure 4.2: B) for 3 days appear wider compared to the untreated cultures

(Figure 4.2: A). TNF treated myotubes (Figure 4.2: C) appeared thinner compared to the

untreated controls (Figure 4.2: A). There were also fewer myotubes in the TNF treated

cultures, compared to the untreated controls (Figure 4.2: C and A respectively); this

suggests that the TNF may have been toxic and killed some myotubes.


60

Figure 4.2: Immunofluorescent microscopy images of day 10 C2C12 myotubes treated with IGF-1 or TNF for 3 days. Representative photomicrographs of myotubes stained with anti-desmin antibodies (in green). Myotubes treated with IGF-1 (10 ng/ml) (B) appeared wider compared to untreated controls (A). In contrast, myotubes treated with TNF (20 ng/ml) (C) show decreased width. Magnification: 10x. Bar: 100 µm.

Quantitative analysis (Figure 4.3) confirmed the various differences in myotube widths

resulting from treatment with either IGF-1 (10 ng/ml) or TNF (20 ng/ml) for 3 days

(Figure 4.3). Myotube widths were quantitated from measurements collected from 50

randomly selected myotubes per culture dish, resulting in totals of 1200, 300 and 900

myotubes being measured for the untreated controls, IGF-1, and TNF treatments

respectively. Myotube widths were taken approximately 50 µm apart throughout the

entire length of the myotube (see Methods Section 2.4.3). IGF-1 treatment induced a

significant increase (P < 0.0001) in the mean myotube width, whereas TNF treated

myotubes had significantly smaller (P < 0.0001) mean widths (decrease of

approximately 25%) compared to the untreated controls (Figure 4.3).


61

Figure 4.3: Quantitative analysis of mean myotube widths of untreated (control), IGF-1 and TNF treated C2C12 cultures. C2C12 myotubes treated for 3 days with IGF-1 (10 ng/ml) show a significant increase in myotube width whereas treatment with TNF (20 ng/ml) induced a significant decrease (to mean myotube width when compared to Untreated (control) myotubes. Results are mean myotube widths + SE of independent experiments indicated for each treatment (n = 50 myotube diameters/experiment). ***P < 0.0001 vs untreated controls.

4.3 Effects of combined treatment with IGF-1 and TNF on day 7 mouse C2C12

muscle myotubes

C2C12 myotubes differentiated for 7 days in the low serum medium received various

combined treatments with IGF-1 and TNF for 3 days. To determine if IGF-1 could

prevent or reverse the atrophic effects of TNF, or if TNF could prevent IGF-1 induced

hypertrophy, the combined treatments were divided into three test groups: 1)

Simultaneous IGF-1/TNF treatment, where both IGF-1 and TNF were administered to

the myotubes at the same time; 2) IGF-1 added before TNF, where myotubes were pre-

treated with IGF-1 for 1 h before TNF treatment; and 3) TNF added before IGF-1 where

TNF was administered 24 h before IGF-1 treatment.


62

Figure 4.4: Quantitative analysis of myotube widths of untreated (control), IGF-1, TNF and combined IGF-1 and TNF treated C2C12 cultures. Combined treatments resulted in no significant change to mean myotube width when compared to untreated controls. Results are mean myotube widths + SE of independent experiments indicated for each treatment (n = 50 myotube diameters/experiment).***P < 0.0001 vs untreated controls. •P < 0.05 vs TNF.

The combination treatments were found to have no significant effect on mean myotube

width when compared to the untreated control cultures (Figure 4.4). All of the combined

treated groups were significantly smaller when compared to the IGF-1 only treated

myotubes, and were larger when compared to the TNF only treated myotubes (Figure

4.4). Although myotubes treated with IGF-1 for 1 h before the administration of TNF

visually appeared to have a greater width compared with the other treatment

combinations, and with the TNF treated myotubes, the difference between them was not

statistically significant.

4.4 Effects of TNF on control FVB and primary cultures of transgenic IGF:C2

mouse myotubes

4.4.1 Effect of TNF on day 4 FVB primary mouse myotubes

The morphological effect of TNF was further tested in primary myotube cultures

derived from 4-6 week old FVB mice. FVB mice were used as the control strain, instead

of IGF:C2 transgenic negative littermates, because FVB mice are the background strain

for the transgenic IGF:C2. The isolation of muscle cells from the FVB mice minimised

the possibility of the inclusion of transgene positive cells, an error that may have


63

occurred if transgenic negative littermates were used as controls. In addition, the

treatment was administered to all primary muscle cell cultures on day 4 of fusion,

compared to day 7 for the C2C12 experiments. This change in treatment time reflected

the accelerated rate at which the primary muscle cells fused into myotubes; at day 4 of

differentiation, myotubes in the primary muscle cell cultures resemble those seen in

C2C12 cultures after 7 days of differentiation. After 3 days of treatment (day 7 after

fusion medium) the myotubes were fixed and stained with desmin antibody and

myotube widths quantified as for the C2C12 experiments.

TNF treated FVB myotubes had significantly smaller (P < 0.05) mean widths

(approximately 20% decrease) when compared to untreated control cultures (Figure

4.5). This TNF induced atrophy was similar to that observed in C2C12 myotubes

(Figure 4.3).

Figure 4.5: Quantitative analysis of myotube widths from TNF atrophy experiments conducted on Wild type FVB and Transgenic IGF:C2 primary skeletal muscle cell cultures. In the control FVB wild-type myotube cultures, TNF treatment alone significantly decreased mean myotube width when compared to untreated FVB wild-type myotubes. The IGF:C2 myotubes cultures were used to test the combined treatment of TNF with IGF-1. In these IGF:C2 myotubes, the addition of TNF had no effect on mean myotube width when compared to untreated IGF:C2 myotubes. Results are mean myotubes width + SE of independent experiments indicated for each treatment (n = 50 myotube diameters/experiment). *P < 0.05 vs untreated FVB wild-type controls.

4.4.2 Testing the effect of TNF on transgenic IGF-1 class 2 primary muscle cells

The combined treatment experiment conducted on the day 7 C2C12 myotubes was

repeated using the primary skeletal muscle cells isolated from transgenic mice that over-

express Class 2 IGF-1 Ea within myotubes and myofibres. The availability of the


64

IGF:C2 transgenic mice provided an opportunity to generate a muscle cell culture model

that did not require the addition of exogenous IGF-1. In this way, the preventative effect

of IGF-1 to TNF induced atrophy could be examined in a primary muscle cell culture.

The addition of TNF (20 ng/ml) to the transgenic IGF-1:C2 day 4 myotubes for 3 days

resulted in no significant change to the mean myotube size when compared to untreated

IGF-1:C2 myotubes. This trend was also observed in the C2C12 myotubes (Section

4.3). Although the myotube widths for the untreated FVB wild-type unexpectedly

appeared visually larger than for the untreated IGF:C2 transgenic myotubes, the

difference between them was not statistically significant.


65

Discussion

The aim of this study was to validate if treatment with IGF-1 to well differentiated day 7

C2C12 myotubes could induce a significant increase in myotube width indicative of

hypertrophy, and if treatment with TNF could induce an atrophic response, specifically

a statistical significant decrease in myotube widths. To quantify the change in mean

myotube widths resulting from the various treatments, it was necessary to develop a

suitable model in which individual myotube widths could be measured, while

concurrently ensuring that the effects on myotube widths were the result of the

treatments acting on the well developed myotubes, and not the result of the activity of

differentiating cells.

With the optimisation of surface coating, cell plating density and fusion medium for

C2C12 myotubes previously determined (Chapter 3), it was necessary to test that the

new C2C12 cells (passage < 5) would serve as a suitable cell culture model. The

importance of maintaining C2C12 cells at low passages (passage < 7) to help preserve

the characteristics of the cell line has been recently emphasised (Fernandez-Celemin et

al. 2002; Dehoux et al. 2007; Hughes et al. 2007). Initial IGF-1 experiments outlined in

Chapter 3 were repeated in the new C2C12 cells. C2C12 cells were cultured in low

serum medium for 7 days before the experimental period. This formed the ‘model

system’ for all experiments conducted on differentiated myotubes in this study. After 7

days of culture in low serum medium, myotubes covered most of the culture and very

few myoblasts remained. This observation was also reported by Tolosa et al (Tolosa et

al. 2005) who investigated the myogenic profile of day 7 C2C12 myotubes, and

confirmed that C2C12 myotubes are more fully differentiated at day 7 (Tolosa et al.

2005). Another study that examined the expression of the structural proteins sarcomeric

actin and myosin at various stages of C2C12 differentiation, revealed that

approximately 5 days after differentiation was induced, and until 10 days when the

study was concluded, C2C12 myotubes express morphological characteristics of adult

skeletal muscle fibres (Burattini et al. 2004).


66

IGF induced myotube hypertrophy

Treatment with 10 ng/ml IGF-1 induced hypertrophy in the day 7 C2C12 myotubes.

This finding confirms the hypertrophic actions of IGF-1 previously published (Rommel

et al. 1999; Rommel et al. 2001; Latres et al. 2005), however in those studies, IGF-1

was administered to cells during differentiation. The present study, however, was

concerned with the effect of IGF-1 on more fully differentiated myotubes, therefore

C2C12 cells were treated on day 7, at which point they are fully differentiated. To

determine the effect of IGF-1 on increasing myotube size (hypertrophy), compared to

increasing the number of myoblasts present (hyperplasia), the time at which IGF-1 is

administered to differentiating cells is critical. Under conditions where fusion is still

occurring at a high rate, C2C12 myoblasts divide within the first 48 h and are not fully

differentiated until day 5-7 (Burattini et al. 2004; Tolosa et al. 2005). Where IGF-1 was

added to C2C12 cells 48 h to 72 h after fusion had been induced (Rommel et al. 1999;

Rommel et al. 2001; Latres et al. 2005), the resulting change in myotube sizes observed

in these cultures after 1-2 days of treatment may have been in part due to fusion of

mononucleated cells into myotubes, in addition to an increase in protein synthesis

within the myotubes. For this reason, the present study purposely cultured C2C12

myotubes to day 7 of differentiation, before IGF-1 was administered (for 3 days), to

specifically determine if IGF-1 was inducing myotube hypertrophy (change to fused

myotube sizes), and not acting on proliferating myoblasts.

TNF induced myotube atrophy

The next series of experiments focused on the effects of the pro-inflammatory cytokine

TNF on myotube morphology, specifically any changes to myotube widths. As for IGF-

1, an independent assay was required to determine that the TNF used in the experiments

was biologically active. Results from the present TNF assay confirmed that the TNF

administered to the cells was biologically active, since C2C12 myoblasts transferred to

low serum medium in the presence of 20 ng/ml TNF did not fuse to form myotubes.

These findings support previous reports that the addition of TNF to differentiating

C2C12 myoblasts inhibits myotube formation in mouse cells (Szalay et al. 1997;

Meadows et al. 2000; Foulstone et al. 2001; Coletti et al. 2002) and also in human

primary muscle cell cultures (Miller et al. 1988; Foulstone et al. 2004).


67

It has been proposed that the inhibitory effect of TNF on myotube formation is the

result of TNF acting through NF-kB activation (Guttridge et al. 2000; Li and Reid 2000)

or by TNF inhibiting the expression of myogenic transcription factors (MyoD and

myogenin) to block the synthesis of messenger RNAs of myogenic differentiation

proteins; specifically skeletal alpha-actin, myosin heavy and light chains (Szalay et al.

1997). In addition, C2C12 myoblasts transferred to low serum in 20 ng/ml TNF undergo

continual cell division compared to cells cultured in low serum alone, which prevents

the G0 cell cycle arrest required for differentiation to occur (Meadows et al. 2000).

It has been shown that TNF appears to have a critical role in skeletal muscle atrophy

and is the key cytokine mediating cachexia (muscle wasting) (Argiles et al. 1997; Glass

2005; Arthur et al. 2008). There have been few studies, however, detailing the

morphological effect of exogenous TNF treatment on cultured C2C12 myotubes, with

no studies examining the induction of myotube atrophy by TNF treatment alone.

Previous the this present study, researchers have developed an in vitro model of skeletal

muscle atrophy using the synthetic, cachectic glucocorticoid Dexamethasome (DEX);

the addition of 10-100 µM DEX for 24 h to C2C12 myotubes 2-4 days post fusion has

been reported to induce a significant decrease to myotube diameters and total protein

content (Stitt et al. 2004; Latres et al. 2005; Sacheck et al. 2004). In muscle, it is known

that DEX activates the Forkhead Box O (FoxO) class of transcription factors, resulting

in increased expression of the muscle atrophy related genes Atrogin-1 (also known as

Muscle Atrophy F-box; MAFbx) and muscle RING finger 1 (MuRF1) (Sandri et al.

2004; Stitt et al. 2004). More recently, a model of C2C12 myotube atrophy has been

reported to be developed though the combined administration of TNF with another pro-

inflammatory cytokine, interferon-γ (IFN- γ) (Dehoux et al. 2007). The present study

was the first to demonstrate that treatment with TNF (20 ng/ml) alone induces

significant changes to myotube widths in both immortalised C2C12 cells (Figure 4.3)

and primary muscle cultures (Figure 4.5).

Li et al (2005) reported that the addition of 6 ng/ml TNF to differentiated C2C12

myotubes induced activation of the Atrogin-1; this has also been shown to occur when

administered at a dose of 5 ng/ml in combination with the inflammatory cytokine,

interferon-γ (IFN- γ) (Dehoux et al. 2007). Combined treatment with TNF/ IFN-γ has


68

also been reported to reduce MyoD and myosin heavy chain (MHC) protein expression

(Guttridge et al. 2000), although TNF alone had no effect on MyoD or MHC, the

authors concluded that effects of TNF on skeletal muscle cellular function could only be

demonstrated when administered in combination with another inflammatory cytokine

(Guttridge et al. 2000). A recent report by Dehoux et al (2007) showed that combined

exogenous treatment with 5 ng/ml TNF/IFN- γ was sufficient to induce myotube

atrophy in differentiated C2C12 myotubes, however the authors did not present data on

the morphological effects of TNF treatment alone. TNF induced atrophy of mature

myotubes in the present study is thus a novel observation and provides a very powerful

new in vitro model.

In the present study, the morphological effects of TNF alone was tested on day 7 C2C12

myotubes, with further confirmation sought from experiments conducted on primary

skeletal muscle cell cultures generated from 5-6 week old FVB mice. A detailed

morphological examination of mean myotube widths from C2C12 (Figure 4.3) and FVB

(Figure 4.5) myotube cultures demonstrated that 20 ng/ml TNF treatment for 3 days was

sufficient to induce a significant decrease in mean myotube width when compared to

untreated control measurements. Those data demonstrate a robust cell culture model of

TNF induced muscle atrophy utilising either well differentiated C2C12 or primary

myotubes, and provides a very useful approach for new atrophy related studies.

The inclusion of FVB primary myotubes provided an additional cell culture model to

test the morphological effects of exogenous TNF treatment, thus avoiding the need to

administer large amount of TNF in vivo to mice. Colleti et al (2005) previously studied

the morphological effects of TNF in vivo using electroporation and gene transfer of an

expression vector encoding the secreted form of murine TNF (mTNF). They compared

type IIb fibre diameters sampled from the quadriceps, tibialis anterior and

gastrocnemius muscles of mTNF and MOCK (empty construct) mice, 5 weeks after

electroporation. The study found that there was a statistically significant decrease in

myofibre cross-sectional area in the mTNF, compared to the MOCK-electroporated

mice for all of the muscles sampled. The results from the present primary FVB muscle

cell culture experiments over 3 days support Colleti et al’s results by similarly

demonstrating TNF induced myotube atrophy in vitro.


69

The final experiments using the combined treatments tested if IGF-1 could prevent or

reverse the atrophic effect of TNF in the day 7 myotubes. Previously, our laboratory has

shown that muscle specific over-expression of insulin-like growth factor-1 (IGF-1) in

transgenic mdx/mIGF-1 mice significantly reduces myofibre necrosis (Shavlakadze et

al. 2004) and that this protective effect may be due to increased protein synthesis in the

muscles. Using cells isolated from the IGF:C2 mice, interactions between TNF and

IGF-1 signalling pathways were investigated, with preliminary studies focusing on

morphological changes to the IGF:C2 myotubes when treated with TNF. The data (from

C2C12 myotubes) indicate that IGF-1 prevents the myotube atrophy caused by TNF.

These results do not support previously published studies by Dehoux et al (2007), where

the combined treatment of IGF-1/TNF/IFN-γ resulted in a significant decrease in mean

myotube widths when compared to untreated controls. In contrast, the present IGF-

1/TNF experiments prevented both atrophy and hypertrophy and mean myotube widths

were the same as the controls cultures. It is particularly important to note that the results

indicate that it may be possible to reverse TNF induced atrophy, as when IGF-1 was

administered 24 h after TNF treatment was initiated, the myotubes were significantly

wider than those treated by TNF alone; there was no significant difference compared to

untreated controls (Figure 4.4)

The inclusion of muscle cell cultures isolated from transgenic mice that over-express

IGF-1 provided another model to test the combined treatments of IGF-1/TNF. IGF-1

exists as different isoforms due to different exon splicing (Shavlakadze et al. 2005);

IGF-1 isoforms that initiate from exon 2 are termed Class 2 (C2) isoforms. For

transgenic muscle cultures from mice that over-expressed IGF-1 Class 2 (IGF-1:C2),

exogenous treatment with TNF had no effect on mean myotube widths when compared

to untreated IGF-1:C2 myotubes. Importantly, this supports the findings of the C2C12

combined IGF-1/TNF treatment experiments, where IGF-1 prevented TNF-induced

myotube atrophy, and TNF prevented IGF-1 induced hypertrophy.

The collective results from these three experiments using the C2C12 myotubes

targeting; (i) IGF-1 hypertrophy, (ii) TNF atrophy and (iii) combined IGF-1/TNF,

confirm that C2C12 myotubes are a useful cell culture model to test the effects of

exogenous cytokine/growth factor treatments. The primary cell cultures further validate


70

these results. A novel and very important observation was that TNF treatment induced

atrophy in both the C2C12 and FVB primary myotubes. Overall, the results gathered

from all of the experiments confirms the results of those, albeit few, previously

published and also highlights the morphological effects of TNF treatment on cultured

muscle cells.

Chapter Five The role of TNF activated JNK in muscle atrophy in tissue cultured myotubes

71

Chapter Five

The role of TNF activated JNK in

muscle atrophy in tissue cultured myotubes

Introduction

The optimisation of the experimental conditions to study cultured C2C12 myotubes

aged 7 days, for treatment with either IGF-1 or 20 ng/ml TNF for 3 days provides a

valuable and robust system for investigating the morphological response of mouse

myotubes to IGF-1 induced hypertrophy and TNF induced atrophy (see Chapter 4). This

myotube model further elucidated the morphological response to combined treatment

with IGF-1 and TNF and thus emphasised the cross-talk between these different

pathways that can affect myotube width. Using this model, the aims of the following

experiments are to: (i) elucidate the involvement of the TNF activated mitogen-

activated protein kinase (MAPK) family, the c-Jun N-terminal kinases (JNKs), in

situations of TNF induced myotube atrophy and (ii) the impact of TNF activated JNK

on IGF-1 induced myotube hypertrophy. TNF activated JNK signalling is demonstrated

in differentiated C2C12 muscle cells in culture using morphological and phospho-

protein western analysis.

There is now strong evidence that TNF can inhibit IGF-1 expression (Frost et al. 2003)

and abrogate IGF-1 receptor signalling via inhibition of tyrosine phosphorylation of the

IGF-1 receptor docking protein insulin receptor substrate-1 (IRS-1) (Grounds et al.

2008). The inhibitory effect of TNF on IGF-1 signalling appears to be mediated by JNK

phosphorylation of IRS-1 on Serine residue 307 (Ser 307), which reduces the affinity of

IRS-1 for the IGF-1 receptor (Broussard et al. 2003; Strle et al. 2006) (see Section 1.5.3,

Figure 1.4a and b). To determine the extent to which TNF activated JNK signalling is

involved in both mediating myotube atrophy and the inhibition of IGF-1 induced

hypertrophy, JNK inhibitors were used. It was hypothesised that inhibition of TNF

activated JNK would prevent TNF-mediated atrophy and result in TNF treated myotube

sizes resembling those seen in untreated cultures.


72

Initial phospho-protein western blot experiments performed in this study specifically

aimed to determine if treatment with TNF could induce phosphorylation (activation) of

JNK in day 7 C2C12 myotubes. After optimising the most effective minimal dose at

which TNF induces JNK activation, and determining the appropriate time point at

which JNK activity was at a maximum after TNF stimulation, the specific role of JNK

was further investigated using JNK inhibitors. Three inhibitors were assessed for

biological toxicity in C2C12 myotubes, with only one (TAT-TIJIP) appearing to be

most suitable for use in the C2C12 myotubes when applied for 3 days at a 10 µM dose.

Using this inhibitor, the direct activity of the TNF activated JNK signalling pathway

was investigated, with the aim of determining if inhibition of the JNK pathway would

prevent TNF induced C2C12 myotube atrophy. These experiments were then further

validated in FVB and IGF-1:C2 primary myotube cultures.

The JNK inhibitor TAT-TIJIP was shown to prevent TNF induced atrophy in both the

C2C12 myotubes and primary muscle cells. Following this result, the next stage of

experiments sought to confirm that TAT-TIJIP was specifically acting on JNK. To test

this, the activity of the JNK downstream target c-Jun was assessed in TAT-TIJIP treated

cells. JNK binds to the amino-terminal region of the transcription factor c-Jun and

phosphorylates c-Jun at Ser63/73 (Cheng and Feldman 1998; Derijard et al. 1994) (see

Section 1.5.2), resulting in localisation of phosphorylated c-Jun from the cytoplasm to

the nucleus. Immunohistochemical studies with antibodies that detect phosphorylated c-

Jun confirmed the nuclear localisation of c-Jun following stimulation with 20 ng/ml

TNF in day 7 C2C12 myotubes. The nuclear localisation of c-Jun was then further

investigated in day 10 myotubes that were pre-treated with the TAT-TIJIP before

receiving TNF treatment for 3 days. In these studies, the nuclear localisation of

phosphorylated c-Jun was not detected (in contrast with controls) indicating that TAT-

TIJP was effectively inhibiting JNK activity (Cheng and Feldman 1998; Derijard et al.

1994).


73

Results

5.1 TNF activates the JNK signalling pathway in C2C12 myotubes

5.1.1 Optimising conditions for detecting TNF activation of the JNK signalling

pathway in day 7 C2C12 myotubes

To determine whether JNK is phosphorylated in response to TNF treatment, and at what

time point this activation is at its maximum, differentiated day 7 C2C12 myotubes were

stimulated with 20 ng/ml TNF for different time intervals, and lysates were probed for

phospho-JNK levels (Figure 5.1: A). To ensure that the solvent (sterile ddH2O) in which

TNF was dissolved had no effect on activation of JNK, an equal volume (1 µl) of

ddH2O was added to the ‘untreated’ (no TNF treatment) cultures. Following Cuenda

and Cohen's (1999) report that 10 µg/ml of anisomycin administered for 30 mins to day

6 C2C12 myotubes resulted in a four-fold activation of JNK, treatment with 10ug/ml of

the protein synthesis inhibitor Anisomycin was included as a positive control in the

study.

Initial time course experiments involved removing the cells from the incubator (which

was maintained at a constant 37ºC) and moving them to the adjacent laminar flow hood,

thereby ensuring that sterile conditions were maintained whilst treatment was

administered. By following this process, the cells were exposed to changes in

temperature, ranging from 37ºC, to room temperature (approximately 23ºC-25ºC), and

then being placed back at 37ºC for the remaining time. This also resulted in some

movement of the fluid in the culture dishes. Western blotting studies detected high

levels of JNK phosphorylation in most of the untreated control lysates, as well as in the

TNF and anisomycin (positive) control lysates. It is hypothesised that the background

phosphorylation of JNK in these controls was due to stress resulting from both

temperature change in and the mechanical disturbance of the cultures. A modified

procedure of administering TNF to undisturbed cultures within the incubator was thus

used for the final experiments (see below). The highest level of JNK phosphorylation

was detected in lysates extracted from myotubes that had been stimulated with 10 µg/ml

Anisomycin for 15 min (Figure 5.1: A). The total levels of JNK protein remained

unchanged in the untreated, TNF and the Anisomycin treated lysates (Figure 5.1: B).


74

It is important to note that the phosphorylated JNK (phospho-JNK) and total JNK

antibodies used in this study detect both JNK1/2, and thus two bands are shown in

Figure 5.1 A and Figure 5.2 A. Sometimes, a third band was also visible, with a

molecular weight of approximately 44kDa as shown in the phospho-JNK blots (Figures

5.2 and 5.3: A) and total JNK blots (Figures 5.1 and 5.2: B). This additional band was

also observed when the same antibodies (from the same manufacturer, Cell Signaling

Technologies) were applied to cell lysates that had been extracted from C2C12

myotubes, but did not appear in lysates that had been extracted from proliferating

C2C12 myoblasts as described in Huang et al. (2007).

Figure 5.1: Optimising conditions for detecting TNF phosphorylation of JNK in differentiated (day 7) C2C12 myotubes: preliminary experiments Myotubes cultured in fusion (low serum) medium for 7 days were treated at room temperature with 1 µl of solvent (sterile ddH2O) to serve as the untreated controls or 20 ng/ml TNF for different time intervals: 0 min (untreated control), 5 min, 10 min, 15 min and 30 min. Myotubes were stimulated with 10 µg/ml Anisomycin for 15 min to serve as a positive control for JNK phosphorylation. Cell lysates were extracted and immunoblotted with (A) anti-phospho-JNK and (B) total JNK antibodies. (A) Phosphorylation of the JNK1 and JNK2 was detected in the untreated (ddH20) controls, TNF treated and Anisomycin treated lysates. (B) Equal loading was monitored with total JNK antibody. There was no change in the level of total JNK1 and JNK2 detected between the 3 treatment groups. It was concluded that phosphorylation of JNK in the controls was a stress response to the initial treatment procedure.

This method of cell stimulation was unsuitable due to the high levels of phospho-JNK

in the untreated (sterile ddH2O control) lysates. To reduce the stress incurred on the

cells when being removed from the incubator to be treated, a change was made to the

way the treatment protocol and cells the were carefully stimulated inside the incubator,

thus avoiding the cells being exposed to sudden changes in temperature and movement

of the culture medium. This revised treatment protocol was successful negated.


75

Western blot analysis of the untreated control lysates from the second set of

experiments showed no detectable phospho-JNK in the untreated controls (Figure 5.2:

A). In the TNF treated myotube lysates, there was initial phosphorylation of JNK within

10 min of TNF stimulation, with the highest level of JNK phosphorylation detected

within 15 min (Figure 5.2: A). This phosphorylation of JNK then declined but persisted

for up to 30 min (Figure 5.2: A). Anisomycin stimulated a higher level of JNK2 (54

kDa) phosphorylation compared to that seen at 15 min in the TNF treated lysates.

Conversely, 15 min stimulation with TNF induced a higher level of JNK1 (46 kDa)

phosphorylation than for the Anisomycin treated lysates (Figure 5.2: A).

Figure 5.2: Time course assay of TNF phosphorylation of JNK in differentiated (day 7) C2C12 myotubes: final protocol. (A) Western blot of lysates immunoblotted with anti-phospho-JNK shows that in myotubes cultured in differentiation (low serum) medium for 7 days and treated at 37ºC with 20 ng/ml TNF, initial phosphorylation of JNK is detected within 10 min with the highest level of phosphorylation seen within 15 min. Very low levels of JNK phosphorylation are seen up until 30 min after stimulation. JNK phosphorylation was not detected in the untreated controls. Anisomycin, at a dose of 10 µg/ml, was administered for 15 min to serve as a positive control for JNK phosphorylation. (B) Equal loading was monitored with total JNK antibody. 5.1.2 Titration study to determine if JNK phosphorylation is dependant on dosage of TNF To determine whether the phosphorylation of JNK detected within 15 min of

stimulation with 20 ng/ml TNF was the highest level that could be induced in the day 7

myotubes, and additionally determine if higher dosages of TNF treatment would sustain

JNK activation, the cells were treated with varying dosages of TNF and protein was

extracted at 15 and 30 min.


76

In the day 7 myotubes, western blot analysis showed no detectable differences in levels

of JNK activation induced by treatment with 20 ng/ml or 50 ng/ml TNF within 15 min.

The lowest level of JNK phosphorylation was observed in the lysates extracted from

myotubes that had received 100 ng/ml TNF (Figure 5.2: A). There was a slightly higher

level of JNK phosphorylation at 30 min in the 50 ng/ml TNF treated lysates, compared

to the level observed at 30 min in the 20 ng/ml and 100 ng/ml TNF treated samples

(Figure 5.3: A). Phosphorylation of JNK was not detected in the untreated controls

(Figure 5.2: A).

Figure 5.3: Titration study to determine the minimum effective dose of TNF to induce phosphorylation of JNK in differentiated (day 7) C2C12 myotubes. (A) Western blot of lysates immunoblotted with anti-phospho-JNK shows that treatment of 7 day myotubes with 20 ng/ml, 50 ng/ml or 100 ng/ml TNF induced comparable levels of JNK phosphorylation. Phosphorylation of JNK was not detected in the untreated controls. Anisomycin, at a dose of 10 µg/ml, was administered for 15 min to serve as a positive control for JNK phosphorylation. (B) Equal loading was monitored with total JNK antibody.

5.2 JNK inhibitors used in TNF induced myotube atrophy studies

5.2.1 Selection of a suitable JNK inhibitor: evaluation of three different inhibitors

at two dosage levels in C2C12 myotubes

After confirming that exogenous treatment with 20 ng/ml TNF for 15 min was sufficient

to induce activation of the JNK signalling pathway (as demonstrated by western blot

analysis – see above), the next step in the present study was to determine which JNK

inhibitors were most suitable (i.e.; non toxic) for use in conjunction with the TNF

induced muscle cell atrophy model (as described previously - see Section 4.2).


77

The toxicity of three JNK inhibitors was tested (TAT-TIJIP, SP600125 and AS601245)

in conjunction with their respective controls; TAT peptide (control peptide for TAT-

TIJIP) and DMSO (diluent for TAT-TIJIP and TAT); see Section 1.5.4. Of the three

inhibitors tested, TAT-TIJIP is a novel ATP-non-competitive, JIP-derived cell

permeable peptide inhibitor of JNK, produced by Dr Marie Bogoyevich (Arthur et al.

2007). The ATP-competitive inhibitors (SP600125 and AS601245) are commercially

available. Each inhibitor was tested at two dosages (5 µM and 10 µM) for 3 days to

determine the dose at which the inhibitor did not have an adverse effect on myotube

morphology. On day 3 of treatment the myotubes were fixed and any changes to

myotube morphology were visually assessed (see Section 2.4.3).

On day 3 of treatment (day 10 after fusion) the phase contrast images of ‘control’

C2C12 myotubes (those treated with the TAT peptide or with DMSO) showed no

difference in appearance when compared to untreated myotube cultures (Figure 5.4: A,

B and C).

The myotubes that had been treated for 3 days with either 5 µM or 10 µM of the novel

JNK inhibitor peptide TAT-TIJIP had a similar appearance to the untreated and control

cultures (Figure 5.4: D1 and D2). The myotubes treated with of 5 µM of SP600125 also

appeared morphologically normal (Figure 5.4: E1), however at the higher dose (10 µM)

the myotubes appeared slightly thinner, with a noticeable increase in the number of

unfused/dying cells (Figure 5.4: E2). The myotubes that had received 5 µM AS601245

appeared thinner than the untreated cultures and were starting to detach from the culture

dish after 3 days of treatment; this suggests that the inhibitor was toxic (Figure 5.4: F1).

At the higher dose (10 µM) AS601245 was found to be highly toxic, with considerable

cell death resulting in very few myotubes remaining alive (Figure 5.4: F2).


78

Figure 5.4: Toxicity evaluation of three JNK inhibitors on day 10 C2C12 myotubes. Phase contrast images show untreated C2C12 myotubes at day 10 of culture in fusion medium (A1 and A2), control myotubes that had been treated for 3 days with DMSO (B1 and B2), TAT peptide treated control myotubes (C1 and C2) and test myotubes that had been treated with the JNK inhibitors TAT-TIJIP (D1,2), SP600125 (E1,2) or AS601245 (F1 and F2). Myotubes treated with 5 µM and 10 µM TAT-TIJIP (D1 and D2 respectively) showed no difference in appearance compared to untreated (A1 and A2) and TAT treated controls cultures (C1 and C2). SP600125 was non-toxic to C2C12 myotubes at a dose of 5 µM (E1) but had an adverse effect on myotubes at a dose of 10 µM (E2). Both doses of AS601245 were toxic to C2C12 myotubes (F1 and F2). Magnification: 10x. Bar: 100 µm.


79

5.2.2 Experiments to determine if TNF induced myotube atrophy can be prevented

by pre-treatment with the JNK inhibitor TAT-TIJIP

The following experiments were performed using C2C12 myotubes and primary

myotube cultures. It is important to note that the experiments involving the primary

FVB muscle cells were not conducted in direct parallel (at the same time) as those

experiments involving the IGF:C2 muscle cell cultures. For this reason, the results from

experiments using the control FVB muscle cells are not directly compared to those

generated from the IGF:C2 muscle cell cultures.

5.2.2.1 Analysis of the morphological effects of TAT-TIJIP on untreated and TNF

treated C2C12 myotubes

To explore the involvement of JNK in mediating TNF induced myotube atrophy, day 7

C2C12 myotubes were pre-treated with TAT-TIJIP (10 µM) for 1 h before treatment

with TNF for 3 days. Quantitative analysis (Figure 5.5) confirmed highly significant

differences in myotube widths resulting from treatment with TNF (20 ng/ml) alone

compared to combined TAT-TIJIP/TNF treatment for 3 days. Myotube widths were

quantified as previously outlined (see Section 2.4.3). Width measurements were

collected from 50 randomly selected myotubes from a number of cultures dishes; the

number of repeats is specified for each group in Figure 5.5. As a reference, the results

for the untreated and TNF treated C2C12 myotubes (shown in Figure 4.3), were

included in this study to compare pre-treated combined TAT-TIJIP/TNF treated (TAT-

TIJIP + TNF) myotubes with TNF only treated. As there were no significant differences

between new controls (n = 5) and old controls (n = 6), the results were pooled together.

Pre-treatment with TAT-TIJIP (10 µM) 1 h before the administration of TNF (20 ng/ml)

resulted in a significant increase (P < 0.0001) in mean myotube width when compared

to untreated (~20% difference) and TNF treated (~55% difference) control myotubes

(Figure 5.5). The myotubes treated with TAT-TIJIP + TNF were also significantly

wider (P < 0.01) than those that had been treated with TAT-TIJIP alone (Figure 5.5).

Both TAT-TIJIP and the TAT control peptide had no effect on myotube widths when

compared to the untreated controls (Figure 5.5). Combined treatment with the TAT

control peptide and TNF (TAT control + TNF) resulted in a significant decrease (P <

0.0001) in mean myotube widths, when compared to untreated, TAT-TIJIP and TAT


80

control treated myotubes; conversely, when compared to TNF treated myotubes, no

significant difference in mean myotube widths was evident (Figure 5.5).

Figure 5.5: Mean width of untreated control C2C12 myotubes and C2C12 myotubes treated with TNF alone, TAT-TIJIP alone, combination of TAT-TIJIP and TNF, TAT alone and combination of TAT and TNF. C2C12 myotubes treated for 3 days with TNF (20 ng/ml) show a significant decrease in myotube width, whereas pre-treatment with the JNK inhibitor TAT-TIJIP for 1 h before TNF treatment induced a significant increase to mean myotube width when compared to Untreated (control) myotubes. Treatment with TAT-TIJIP alone or TAT control alone or in combination with TNF had no effect on the mean myotube width when compared to untreated (control) myotubes. Results are mean myotube widths + SE of independent experiments indicated for each treatment (n = 50 myotube diameters/experiment). *P < 0.01 and *P < 0.0001. The numbers within each bar represent the number of times the experiments were repeated in separate dishes, with the results pooled.


treated control FVB mouse myotubes in primary cultures

To further test the preventive effects of TAT-TIJIP on TNF induced atrophy, primary

myotube cultures derived from 4-6 week old FVB mice were generated and treated with

combined TAT-TIJIP + TNF. As in the C2C12 myotubes, pre-treatment with TAT-

TIJIP 1 h before treatment with TNF (TAT-TIJIP + TNF group) for 3 days resulted in a

significant increase (P < 0.0001) in mean myotube widths when compared to TNF

treated (28% difference) control myotubes (Figure 5.6). The myotubes treated with

TAT-TIJIP + TNF were also show a significant increase in width (P < 0.005) compared

to those treated with TAT-TIJIP alone; this is similar to the results from the analysis of

C2C12 myotubes. Again, when administered alone as controls, both TAT-TIJIP and the


81

TAT control peptide had no effect on myotube widths, in comparison to the untreated

controls (Figure 5.6).

The combined treatment with the TAT control peptide and TNF (TAT control + TNF)

resulted in a significant decrease (P < 0.05) in the mean width of the FVB myotubes in

comparison to the untreated TAT-TIJIP and TAT control treated myotubes. Also, when

compared to the TNF treated myotubes, no significant difference in mean myotube

widths was evident (Figure 5.6). These results are again consistent with the initial study

conducted using C2C12 myotubes (Figure 5.5).

Figure 5.6: Mean width of untreated control primary FVB myotubes and FVB myotubes treated with TNF alone, TAT-TIJIP alone, combination of TAT-TIJIP and TNF, TAT alone and combination of TAT and TNF. FVB primary myotubes treated for 3 days with TNF (20 ng/ml) show a significant decrease in myotube width, whereas pre-treatment with the JNK inhibitor TAT-TIJIP for 1 h before TNF treatment induced a significant increase to mean myotube width when compared to TNF alone treated myotubes. Treatment with TAT-TIJIP alone or TAT alone had no effect on mean myotube width when compared to untreated (control) myotubes. Significant differences between the mean myotube widths are seen when myotubes treated with TAT-TIJIP alone are compared to those that had received combined TAT-TIJIP + TNF treatment, with the latter showing an increase in mean myotube widths. Results are mean myotube widths + SE of independent experiments indicated for each treatment (n = 50 myotube diameters/experiment). *P < 0.05, **P < 0.005 and *P < 0.0001. Data were pooled form three independent experiments


treated transgenic IGF:C2 mouse myotubes in primary cultures

The combined TAT-TIJIP + TNF treatment experiments conducted on the day 7

C2C12, myotubes and day 4 FVB, myotubes were repeated using primary cultures of

myotubes from IGF:C2 transgenic mice. The IGF:C2 myotubes provided an opportunity

to test whether the preventative effect of (endogenous) elevated IGF-1 on TNF induced


82

myotube atrophy (see Figure 4.5) could be further enhanced when (TNF) activated JNK

is inhibited by TAT-TIJIP.

The addition of TNF (20 ng/ml) to the IGF:C2 day 4 myotubes for 3 days resulted in no

significant change to mean myotube size in comparison to the untreated IGF:C2

myotubes (see Section 4.4.2). Pre-treatment with TAT-TIJIP (10 µM) at 1 h before the

administration of TNF (20 ng/ml) significantly increased (P < 0.05) mean myotube

widths compared to the TNF treated (~ 20% difference) and TAT control + TNF (~25%

difference) control IGF-1:C2 myotubes (Figure 5.7).

In contrast to the results using C2C12 (Figure 5.5) and FVB myotubes (Figure 5.6),

transgenic IGF:C2 myotubes treated with TAT-TIJIP + TNF presented no significant

increase in myotube widths compared to the controls treated with TAT-TIJIP alone.

Further, both the TAT-TIJIP and the TAT control peptide had no effect on myotube

widths compared to untreated controls (Figure 5.7) as was also observed for both

C2C12 and FVB cultures. Surprisingly, the IGF:C2 myotubes treated with the TAT

control alone appeared to have a larger width compared to the untreated and TAT-TIJIP

only treated myotubes, although this difference was not statistically significant. IGF:C2

myotubes treated with TAT control + TNF, were significantly smaller in width (P <

0.05; ~20%) compared with control TAT only treated myotubes and were actually the

same size as myotubes treated with only TNF.


83

Figure 5.7: Mean width of untreated control primary IGF:C2 myotubes and IGF:C2 myotubes treated with TNF alone, TAT-TIJIP alone, combination of TAT-TIJIP and TNF, TAT alone and combination of TAT and TNF. The transgenic IGF:C2 primary myotubes that were treated for 3 days with TNF (20 ng/ml) alone show no changes to mean myotube width when compared to untreated myotubes. Treatment with TAT-TIJIP alone or TAT alone had no effect on mean myotube width when compared to untreated (control) myotubes. Myotubes that had received combined TAT-TIJIP + TNF treatment are significantly wider when compared to those that had received TNF treatment alone. Myotubes that had been treated with TAT control alone were also significantly wider than those that had received combined TAT + TNF treatment. (n = 50 myotube diameters/experiment). *P < 0.05. Data were pooled form 3 independent experiments

5.3 Confirmation of the inhibition of JNK phosphorylation by TAT-TIJIP:

immunofluorescent nuclear localisation of the transcription factor c-Jun

The inhibitory effect of TAT-TIJIP on JNK was verified by assaying the nuclear

localisation of phosphorylated c-Jun (a downstream target of phosphorylated JNK) in

C2C12 and primary muscle cell cultures. Since the phosphorylation of JNK results in

the localisation of phosphorylated c-Jun, the inhibition of JNK phosphorylation would

be expected to prevent nuclear localisation of phosphorylated c-Jun (Melino et al.

2008).

The first immunonuclear localisation bioassay was performed to determine whether

treatment with TNF (20 ng/ml) induced the phosphorylation and nuclear localisation of

c-Jun in well differentiated day 7 C2C12 myotubes (see Section 5.3.1). Following this,

the inhibition of JNK phosphorylation by TAT-TIJIP was tested by analysing of nuclear

located phosphorylated c-Jun (see Section 5.3.2). The later study was performed in all


84

three skeletal muscle cell cultures, immortalised C2C12 myotubes and primary muscle

cultures generated from FVB and transgenic IGF:C2 mice.

5.3.1 Time course analysis of the nuclear localisation of phosphorylated c-Jun in

day 7 C2C12 myotubes

To determine the involvement of phosphorylated JNK in inducing the nuclear

localisation and phosphorylation of c-Jun, in mediating TNF induced myotube atrophy,

a time course of c-Jun activity was performed in day 7 C2C12 myotubes using

immunofluorescent-labelled antibodies that detect phosphorated c-Jun at Serine 73

(Ser73). It is well documented that JNK1 binds to the amino-terminal region of c-Jun

and phosphorylates c-Jun at two sites (Serine 63 and Serine 73) activating c-Jun-

dependent transcription (Derijard et al. 1994; Kyriakis and Avruch 2001). Strle et al

(2006) previously described the direct relationship between TNF phosphorylated JNK

and c-Jun in C2C12 myoblasts, reporting that phosphorylation of c-Jun was detectable

by western blot as early as 5 min after stimulation with TNF (1 µg/ml).

Day 7 C2C12 myotubes were stimulated with TNF (20 ng/ml) and fixed at various

times (15 min, 30 min, 1 h and 2 h) after treatment. The myotubes were then double

immunostained for phosphorylated c-Jun and desmin. The immunofluorescent

microscopy images shown in Figure 5.8 are representative overlays of 3 images taken at

various excitation wavelengths, depending on the detection reagent for the secondary

antibodies; anti-phosphorylated c-Jun was detected at an excitation of 593 nm

(visualised red) and anti-desmin at 495 nm (visualised green). The Hoechst nuclear stain

(Hoechst 33342) was visualised blue (at 357 nm). Therefore, when the 3 images are

overlayed, the translocation of activated c-Jun appears purple in colour.

The nuclear localisation of phosphorylated c-Jun was assessed visually in the myotubes.

Images of day 7 C2C12 myotubes that were stimulated with TNF (20 ng/ml) showed

nuclear localisation of phosphorylated c-Jun within 15 min after treatment, with the

phosphorylation persisting for up to 2 h (Figure 5.8). In the TNF treated C2C12

cultures, nuclear localisation of phosphorylated c-Jun was also detected in cells that did

not stain positive for desmin (Figure 5.8). The untreated cultures (those exposed to

ddH20 the diluent for TNF) had no detectable phosphorylated c-Jun (Figure 5.8).


85

Figu

re 5

.8:

Exp

osur

e to

TN

F in

crea

ses

phos

phor

ylat

ed c

-Jun

in C

2C12

myo

tube

s. M

yotu

bes

wer

e ei

ther

(A

) U

ntre

ated

(co

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l) or

(B

) ex

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d to

20

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l TN

F (1

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ith a

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c-Ju

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. The

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M

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20x

. Sca

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00µm


86

5.3.2 Immunofluorescent microscopy analysis of TAT-TIJIP inhibition of c-Jun

nuclear localisation (phosphorylation) in TNF induced myotube atrophy in C2C12,

FVB and IGF:C2 cultures

The nuclear localisation of phosphorylated c-Jun was also assessed visually in all

myotube cultures. Cultures were pre-treated with TAT-TIJIP for 1 h before TNF (20

ng/ml) was administered for 3 days (following the protocol for the morphological

studies in Section 2.4.2). The overlayed immunofluorescence microscopy images are

presented in Figure 5.9. In the untreated cultures (exposed to only ddH20 the diluent for

TNF) phosphorylation and nuclear localisation of c-Jun was not detected in the desmin

positive myotubes (Figure 5.9: A, B and C) but was detected in the TNF treated cultures

(Figure 5.9: A2, B2 and C2, indicated by arrows). In some cultures, the nuclear

localisation of phosphorylated c-Jun was also detected in cells that did not stain positive

for desmin (Fig 5.9: A2, B2, C2, C3 and C4, indicated by the asterisk).


87

Figu

re 5

.9: T

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88

5.4 Analysis of IRS-1 and AKT (downstream of IGF-1) to further investigate TNF

inhibition of IGF-1 signalling via JNK, in the model of C2C12 myotube atrophy

It has been suggested that activated JNK is involved in abrogating IGF-1 signalling, via

phosphorylation of the adaptor molecule IRS-1 on serine 307 to disrupt its association

with the IGF-1 receptor (reviewed in Grounds et al. 2008), thus preventing activation

(phosphorylation) of downstream AKT (see Section 1.5.3, Figure 1.4a). To investigate

the cross-talk between TNF and IGF-1 signalling in C2C12 myotubes, a western

analysis was used to measure activation (phosphorylation) of two signalling events

downstream of the activated IGF-1 receptor, specifically the phosphorylation of IRS-1

and AKT.

5.4.1. Optimising western blotting conditions for immunoprecipitated IRS-1 from

C2C12 day 7 myotubes treated with IGF-1

As it has been previously reported that 15 min treatment with IGF-1 stimulates tyrosine

phosphorylation of IRS-1 in C2C12 myoblasts (Milasincic et al. 1996; Strle et al. 2006),

initial optimising phospho-protein experiments were performed as a time course assay

on untreated or IGF-1 (10 ng/ml) treated (5 min, 15 min, 30 min, 60 min and 1 h)

C2C12 day 7 myotubes, Immunoprecipitation of phosphorylated proteins from cell

culture or muscle samples has not been previously performed within the Grounds

laboratory, despite numerous repeated attempts over three years, which included:

trailing and testing a range of protein extraction buffers; implementation of various

modifications to the immunoprecipitation protocol (these include: incubating the protein

beads with the lysates anywhere from 2-12 hrs, with/without rocking); testing different

protein agarose beads (Protein A or Protein G) to determine if the beads were not

capturing the IRS-1 antibodies; and the purchasing and testing various IRS-1 antibodies

from various antibody companies (in an attempt to determine if the antibody was the

problem). Despite these attempts, this assay was not successful in detecting tyrosine

phosphorylated IRS-1 in C2C12 myotubes stimulated with IGF-1 or 100nM Insulin (to

act as a positive control - del Aguila et al. (1999) for 15 min. Therefore, advice was

sought from Dr Jon Whitehead, Head, Cell Signalling Group at the Diamantina Institute

for Cancer, Immunology and Metabolic Medicine, Princess Alexandra Hospital,

University of Queensland, as he has successfully used this assay in published studies on


89

both 3T3 adipocytes and Chinese Hamster Ovary (CHO) cells (Thomas et al. 2006).

Accordingly, I visited his laboratory for one intensive week of laboratory work in

September, 2008

At Dr Whitehead’s laboratory I had access to a number of positive controls which I

could run in parallel to test (Grounds) lysates. The positive controls were lysates

extracted from C2C12 myotube that had been stimulated with Insulin (100nM) for 15

min (del Aguila et al. 1999), in addition to lysates from untreated and Insulin treated (15

min) CHO cells over expressing the insulin receptor (IR) and IRS-1 (IRS.1) (Clark et al.

1998).

The assay was performed under Dr Whitehead’s supervision. It again, however, proved

unsuccessful with no tyrosine phosphorylation (pY) of IRS-1 detected in our C2C12

myotubes stimulated with 100nM Insulin (15 min) compared to the positive controls

from Dr Whitehead’s laboratory (15 min Insulin stimulated C2C12 myotubes and CHO-

IR-IRS-1 cells). The results from the assay thus suggest that the C2C12 cells that were

used throughout this PhD research project had very low levels of IRS-1 that could not

be detected using the current immunoprecipitation protocols.


90

. Figure 5.10: Comparison of tyrosine phosphorylation (pY) of IRS-1 in Insulin stimulated C2C12 cells from two different laboratories. The phosphorylation of IRS-1 was detected in lysates extracted from C2C12 myotubes that had been cultured from cell stocks at Dr Whitehead’s Laboratory, and in the positive controls; CHO-IR_IRS-1 stimulated with Insulin. There was no detectable level of pY IRS-1 in our stocks (Grounds). IRS-1 protein is present in our stocks (Grounds) and was detected in all lysates from both laboratories. In the merged image, pY IRS-1 is visualised as a yellow colour and only seen in Insulin stimulated samples form Dr Whitehead’s Laboratory (C2C12: Whitehead and CHO-IR: wt.IRS-1).

5.4.2. Western blotting analysis of AKT from C2C12 day 7 myotubes treated with

IGF-1 or TNF

Due to the problems associated with detecting phosphorylated IRS-1, the

phosphorylation of AKT was studied in C2C12 myotubes that had been treated with

IGF-1. As it has been demonstrated (in day 2 and day 4 C2C12 myotubes) that IGF-1

(10 ng/ml) induces phosphorylation of AKT (a well defined target downstream of

activated IRS-1), analysis of AKT phosphorylation is thus a logical choice to

demonstrate inhibition by TNF of IGF-1 signalling (Rommel et al. 1999; Rommel et al.

2001).

In day 7 C2C12 myotubes treated for 15 min with IGF-1 (10 ng/ml), or TNF (20 ng/ml),

phosphorylated AKT was detected and this level of activation was slightly higher than


91

that detected in the untreated cultures. Densitometry was performed on this one blot and

indicated that treatment with either TNF or IGF-1 induced activation of AKT

(approximately 11% and 18% increase respectively) compared to the level of activation

in the untreated control sample. Total levels of AKT protein remained relatively

unchanged in the IGF-1 and TNF treated and untreated control lysates (Figure 5.11: B).

Figure 5.11: Activation of AKT in differentiated (day 7) C2C12 myotubes treated with TNF or IGF-1. (A) Western blot of lysates immunoblotted with anti-phospho-AKT shows that in myotubes cultured in differentiation (low serum) medium for 7 days and treated at 37ºC with 1 µl of solvent (sterile ddH2O) to serve as the untreated controls, 20 ng/ml TNF or 10 ng/ml IGF-1 for 15 min. The level of AKT phosphorylation was slightly higher in the TNF and IGF-1 treated samples. (B) Equal loading was monitored with total AKT antibody.

Based on the preliminary results of this western blot, phospho-protein analysis of

activated AKT was also proved to be unsuitable assay for demonstrating the cross talk

between IGF-1 and TNF signalling pathways, because of due to the increase in

phosphorylated AKT observed in the TNF samples, compared to the untreated (sterile

ddH2O) controls.


92

Discussion

It has been suggested that activation of the JNK pathway is involved in the

pathophysiology of a number of inflammatory conditions, such as inflammatory bowel

disease (Roy et al. 2008), Duchenne Muscular Dystrophy ( mouse models -

Kolodziejczyk et al. 2001; Lang et al. 2004) and in rheumatoid arthritis ( rat models -

Han et al. 2001). It is also widely considered that TNF plays a critical role in skeletal

muscle atrophy (see Chapter 4), although the precise role of TNF activated JNK in

situations of TNF mediated myofibre atrophy is still largely unknown. Cell signalling

studies have suggested that the damaging effects of TNF may also be the result of JNK

interference with IGF-1 synthesis (Frost et al. 2003) or IGF-1 signalling, through the

association of JNK1 with the IGF-1 docking protein IRS-1 (Broussard et al. 2004;

Broussard et al. 2003; Strle et al. 2006; Grounds et al. 2008).

Strle et al (2006) exemplified the use of the JNK inhibitor peptide SP600125 to

establish that TNF activated JNK suppresses the biological activity of IGF-1 in

proliferating C2C12 myoblasts. The data obtained from the present study extend this

knowledge to both C2C12 myotubes and to primary cultures of mouse skeletal muscle

treated with TNF. In the present series of studies, a novel JNK inhibitor (TAT-TIJIP)

was included to test the role of activated JNK in TNF induced myotube atrophy.

With the aim of elucidating the role of JNK in TNF induced myotube atrophy, the initial

protein analysis experiments were performed to determine whether treatment with TNF

(20 ng/ml) is sufficient to induce JNK activity in well differentiated day 7 C2C12

myotubes. Previously, western blot analysis of TNF induced phospho-JNK activity has

only been shown in proliferating C2C12 myoblasts (Strle et al. 2006; Huang et al. 2007)

or by immunoprecipitation from C2C12 myotubes (at day 4 of fusion) using JNK1

antibody followed by in vitro kinase assay using GST-c-Jun as substrate (Srivastava et

al. 2007). The results presented in this study show for the first time, using western blot

analysis of total cell lysates from mature myotubes, that TNF strongly activates JNK

within 10 to 15 min after the initial TNF stimulation (Figure 5.2).

Previous research investigating the function of JNK has largely depended on the use of

inhibitors. Such JNK inhibitors have been categorised into two groups; chemical


93

inhibitors that are ATP-competitive (i.e. that target the ATP binding site of JNK and

subsequently other MAPK) and newer peptide inhibitors that are ATP-non-competitive

(directly bind to sites other that the ATO-binding site of JNK) (Bogoyevitch 2005 and

see Section 1.5.3, Figure 1.4a and b). To evaluate the most suitable JNK inhibitor for

use in the TNF induced models of skeletal muscle atrophy, the toxicity of two ATP-

competitive inhibitors (SP600125 and AS601245), and one novel ATP-non-competitive

JNK inhibitor (TAT-TIJIP), was tested in C2C12 myotubes.

When applied to C2C12 myotubes for 3 days, both SP600125 (10 µM) and AS601245

(at 5 µM and 10 µM concentration) were toxic in the cell cultures. The toxicity of

SP600125 in C2C12 cells has been previously demonstrated in C2C12 myoblasts when

applied for 24 h at a concentration of 10 µM (Strle et al. 2006). However, SP600125 is

still considered the most widely used of the JNK inhibitors, despite the specificity of

this inhibitor being questioned (Bain et al. 2003; Bogoyevitch and Arthur 2008).

The toxic effect of AS601245 on the C2C12 myotubes was particularly surprising

because numerous studies have reported on the protective effects of AS601245 in vivo,

in rat models of myocardial ischemia (Ferrandi et al. 2004), and cerebral ischemia

(Carboni et al. 2004). This was also demonstrated in gerbil models (Carboni et al.

2007). The first reports using the ATP-non-competitive, cell penetrable JNK inhibitor

(D-JNKI-1) demonstrated that it has some neuroprotective ability in rat models of

cerebral ischemia (Borsello et al. 2003) (See Section 1.5.4.1).

The ATP-non-competitive JNK inhibitor (TAT-TIJIP) demonstrated no adverse effects

in the present study when administered alone at a concentration of 10 µM, to day 7

C2C12 myotubes for 3 days, or when administered in combination with TNF, in both

C2C12 and primary muscle cell cultures. Similarly, a longer D-amino acid peptide (D-

JNKI-1) has been shown to be non-toxic when administered to C2C12 myoblasts for 36

h at a concentration of 10 µM (Strle et al. 2006) One advantage of using an ATP-non-

competitive JNK inhibitor, such as TAT-TIJIP or D-JNKI-1, relates to their specificity

(see Bogoyevitch et al. 2004; 2005)). Similar to D-JNKI-1, TAT-TIJIP also contains a

cell permeable 10-amino acid human immunodeficiency virus-TAT transporter

sequence which facilitates the entry of the inhibitor into cells (Vives et al. 1997).


94

In the present study, the use of the JNK inhibitor (TAT-TIJIP) supports the direct

involvement of activated JNK in tissue cultures models of TNF induced myotube

atrophy. One of the main mechanisms by which TNF induces myofibre atrophy and

myofibre necrosis, is TNF induced activation of NF-ĸβ (Reid and Li 2001; Eley et al.

2008). Recently, it has also been reported that treatment of C2C12 myotubes with TNF

(50 ng/ml) alone or in combination with IFNγ (10 ng/ml), depressed protein synthesis

within 2 h of administration (Eley et al. 2008; Eley et al. 2008). These findings were

suggested to be due to an increased formation of reactive oxygen species (ROS), via the

activation of p38 MAPK (Eley et al. 2008). Furthermore, it is known that ROS also

activates the JNK and p38 MAPK stress activated protein kinase pathways (Arthur et al.

2008), although the role of JNK in TNF induced muscle wasting is not well established.

A number of reports propose that JNK activation directly interacts with IGF-1

dependant events, and there is strong evidence that TNF activated JNK is involved in

the down regulation of IGF-1 mRNA synthesis in C2C12 myoblasts (Frost et al. 2003).

TNF induced JNK activity has also been suggested as being directly involved in

inhibiting the signalling pathways downstream of IGF-1, by binding to the IGF-1

docking protein IRS-1 (see Grounds et al. 2008). In the present study, repeated attempts

to demonstrate JNK interception with IGF-1 activated IRS-1 proved unsuccessful, thus

the focus was shifted onto examining the activity of AKT in the C2C12 muscle cell

cultures.

The results of the present research show a slight activation in phosphorylated AKT

when TNF was administered to the cells for 15 min compared to the level detected in

the untreated controls. In consideration of recent findings, this observation is not so

unusual, as Eley et al (2008) showed that treatment with TNF (50 ng/ml) induced

activation of mTOR in C2C12 myotubes, although this increase was apparently not

statistically significant. However, these results combined with the results of this present

study (Figure 5.11) suggest that the slight activation of mTOR and AKT induced by

TNF may be the result of autophosphorylation activated by oxidative stress (Eley et al.

2008). The involvement of oxidate stress on inducing activation of AKT is yet to be

fully defined and is an important area of potential future research.


95

The results of the present study strongly suggest that JNK is involved in mediating TNF

induced myotube atrophy. The myotube morphology studies showed that pre-treatment

with the JNK inhibitor TAT-TIJIP prevented TNF induced myotube atrophy. Myotubes

treated with TNF alone displayed nuclear localisation and phosphorylation of c-Jun;

those pre-treated with TAT-TIJIP showed significantly fewer (if any) nuclei displaying

phosphorylated c-Jun. The preliminary signalling studies presented here require

significant further investigation, however, it was demonstrated that treatment with TNF

(as low as 20 ng/ml) is sufficient to induce phosphorylation of JNK in day 7 C2C12

myotubes. The IRS-1 and AKT studies could not be continued due to time constraints;

however the findings presented here provide a solid framework from which further

studies can and should be continued. On the basis of this study performed to this point,

it was accordingly decided that the next series of experiments would be performed in

vivo, in order to assess the protective effects of TAT-TIJIP in situations of muscle

necrosis.

Chapter Six Preliminary study to investigate the effects of TAT-TIJIP on reducing exercised induced myofibre necrosis in

vivo in mdx mice

96

Chapter Six

Preliminary study to investigate the effects of TAT-TIJIP on

reducing exercised induced myofibre necrosis in vivo

in mdx mice

Introduction

In vivo studies in dystrophic mdx mice subjected to exercise-induced muscle fibre

(myofibre) cell death (necrosis) were performed to test the effect of pharmacological

inhibition of JNK on the phenotype of dystrophic muscle, with respect to histological

quantitation of myofibre necrosis. All previous experiments have been performed using

tissue culture muscle models of TNF induced myotube atrophy. It was thus necessary to

further test the beneficial effects of the JNK inhibitory peptide TAT-TIJIP in an in vivo

situation of muscle damage and necrosis.

Animal studies were conducted on mdx mice (C57BL/10ScSnmdx/mdx), a well known

animal model for DMD (Partridge 1991). The experiments were performed using mice

6 weeks of age, selected because previous research has demonstrated that this is the

period at which muscle necrosis stabalises (to approximately 6%); three weeks after the

onset of acute muscle necrosis is seen in the limb muscles of mdx mice (Coulton et al.

1988; McGeachie et al. 1993; Grounds and Torrisi 2004; Shavlakadze et al. 2004). As

previously noted in Section 1.3.2.1, these low levels of muscle necrosis make assessing

the effectiveness of therapeutic interventions on reducing myofibre necrosis difficult,

because of this the mice were subjected to voluntary exercise.

Voluntary wheel running is a suitable exercise model for inducing a significant increase

in quadricep muscle necrosis (Archer et al. 2006; Hodgetts et al. 2006; Radley and

Grounds 2006), however for this to occur, mice from each treatment group are ideally

required to run similar distances, so that any changes to the amount of myofibre

necrosis measured in the quadriceps can be compared to the treatment, thus eliminating

the possibility that these differences are a direct result of the varying distances run.


vivo in mdx mice

97

As there is little published data on the in vivo effects of JNK inhibitors, and no

published data on the in vivo effects of TAT-TIJIP, the current experimental design was

based on methodology presented by Kaneto et al. (2004), which describes methodology

for the use of another the JNK inhibitory peptide, JIP-1-HIV-TAT (JNKI-1; similar to

TAT-TIJIP - see Section 1.5.4.2) in inhibiting JNK in vivo. The optimised protocol

described in this study involved 8 week old obese diabetic C57/BL/KsJ-db/db mice

receiving daily, intraperitoneal injections of JIP-1-HIV-TAT (at a dose of 10 mg/ml) for

2 weeks (Kaneto et al. 2004). For the present study, a similar protocol was followed; 6

week old mdx mice received daily, intraperitoneal treatments of TAT-TIJIP (10mg/kg)

for 3 days. The effects of the TAT-TIJIP treatments on exercised induced muscle

necrosis in the mdx mice are described below.


vivo in mdx mice

98

Results

6.1 The effect of JNK inhibitor TAT-TIJIP on reducing myofibre necrosis in 6

week old voluntary exercised mdx mice

6.1.1 Distance run by the mdx mice

As not all mice exercised voluntarily (Table 6.1), it was necessary to select the mice that

had run the greatest distance, as it is hypothesised that these mice would display greater

muscle damage, compared to those that ran less, and thus the effects of TAT-TIJIP

could be more effectively tested.

There was no difference in the average total distance covered over the 48 h by the

untreated mice (2.56 km), when compared to the TAT-TIJIP treated mice (2.59 km),

with (as noted above) some mice not running at all (Table 6.1). This average total

distance run by the mdx mice is significantly less than previous studies of wheel

running that reported distances between 2-6 km per 24 h (Dupont-Versteegden et al.

1994; Radley and Grounds 2006; Wineinger et al. 1998).

Mdx mice were pre-treated with TAT-TIJIP (10 mg/ml) 24 h (day 1) before being

exposed to 48 h of voluntary exercise (day 2 and 3), during which TAT-TIJIP (10

mg/kg) or sterile ddH20 (control) was administered daily until the mice were sacrificed

(day 4). From each group (Untreated and TAT-TIJIP treated), the three mice that

recorded the largest total distance run (km) (Table 6.1) were selected for further

histological analysis.


vivo in mdx mice

99

Table 6.1: The relation between the voluntary distance ran to the amount of exercised induce myofibre necrosis. From the untreated and treated groups, 3 mice with the highest distances recorded were selected for histological analysis of %necrosis in the quadriceps.

Mouse Treatment Distance (km)

Necrosis (% of quadriceps)

UT-1 Untreated 0.20 not sampled UT-2 Untreated 5.75 4.15 UT-3 Untreated 0.01 not sampled UT-4 Untreated 4.70 5.52 UT-5 Untreated 4.73 11.46 UT-6 Untreated 0.00 not sampled

Average 2.56 7.04 TAT-TIJIP -1 TAT-TI JIP 2.25 1.76 TAT-TIJIP -2 TAT-TI JIP 8.00 9.56 TAT-TIJIP -3 TAT-TI JIP 2.00 not sampled TAT-TIJIP -4 TAT-TI JIP 2.10 not sampled TAT-TIJIP -5 TAT-TI JIP 1.90 not sampled TAT-TIJIP -6 TAT-TI JIP 1.63 not sampled TAT-TIJIP -7 TAT-TI JIP 0.00 not sampled TAT-TIJIP -8 TAT-TI JIP 2.83 6.78

Average 2.59 6.03 6.1.2 Histological analysis of the effects of TAT-TIJIP on reducing myofibre

necrosis in voluntary exercised mdx mice

Of the mice that ran, the right quadriceps muscle was sampled and histologically

examined; and the percentage area of muscle necrosis was then calculated (Figure 6.1).

No difference in the amount of myofibre necrosis was observed in the quadriceps of the

TAT-TIJIP treated mice, when compared to the untreated mice (Figure 6.1).


vivo in mdx mice

100

Figure 6.1: Percentage of myofibre necrosis in right quadriceps muscle of untreated and TAT-TIJIP treated voluntary exercised adult mdx mice. Adult 6 week old mdx mice that were administered with the JNK inhibitory peptide TAT-TIJIP (10 mg/kg body weight) 24 h prior to exposure to voluntary exercise, and then treated daily for a further 2 days during the voluntary exercise period show no significant decrease in muscle damage (necrosis) when compared to voluntary exercised mice that had been treated with ddH20 (Untreated controls; diluent for TAT-TIJIP). Results are mean values + SE of independent experiments indicated for each treatment (n = 3).


vivo in mdx mice

101

Discussion

In the present study it is difficult to determine whether it is of therapeutical benefit to

inhibit JNK by way of using pharmacological inhibitions of JNK (such as TAT-TIJIP)

in in vivo situations of muscle damage. As this was the first study to evaluate the

effectiveness of TAT-TIJIP on reducing myofibre necrosis in vivo, it was extremely

difficult to determine the dosage and duration of TAT-TIJIP treatment required to have

an effect when administered to an in vivo model. For this reason, the experimental

design was based on methodology presented in a study that tested the effectiveness of

JIP-1-HIV-TAT (a JNK peptide similar to TAT-TIJIP) on inhibiting JNK activity in

vivo when administered intraperitoneally in C57BL/KsJ-db/db obese diabetic mice

(Kaneto et al. 2004).

It is important to note that Kaneto et al. (2004) treated mice daily for 2 weeks with

10mg/kg JIP-1-HIV-TAT. As time and resources were limited, in the present study this

preliminary in vivo investigation involved 8 mice, treated daily for 3 days, with

10mg/ml TAT-TIJIP. It was envisaged that the histological analyses from these mice

would then serve as preliminary data from which a new experimental design could then

be modified. However, as discussed below, further studies are required before any

conclusions can be drawn as the effectiveness of TAT-TIJIP in inhibiting the activity of

JNK in vivo, and the effect this has on reducing exercised induced muscle necrosis in

mdx mice.

There are currently no published studies detailing the stability or specificity of TAT-

TIJIP when administered in vivo. The specificity of TIJIP (the inhibitory peptide

comment of TAT-TIJIP - see Section 1.5.4.2) has been tested in vitro (Barr et al. 2002)

and in tissue culture studies. Further, there are very few published studies which

describe using the TAT-TIJIP inhibitor. TAT-TIJIP has been applied to cultured BaF3

cells as part of study aimed at evaluating the contribution of intracellular tyrosine

residues of the granulocyte colony-stimulating factor receptor (Kendrick et al. 2004); in

neuronal cultures to determine the effectiveness of TAT-TIJIP in preventing neurotic

cell death following excitotoxic insult (Arthur et al. 2007); and most recently in T

lymphocytes to ascertain the involvement on JNK in activating the transcription factor

AP-1 and mediating the production of Th1 and Th2 cytokines (Melino et al. 2008). For


vivo in mdx mice

102

these tissue culture studies, it is suggested that TAT-TIJIP is specific and has the ability

to inhibit the JNK pathway; however this is yet to be confirmed in vivo.

As this was the first study to test TAT-TIJIP in vivo, the inhibitor was administered for

3 days to voluntary exercised 6 week old mdx mice, as these animals are the most

convenient model for screening potential compounds for treating patients with severe

muscle wasting such as that seen in DMD (Granchelli et al. 2000; Payne et al. 2006).

Although a functional form of the dystrophin protein is absent in the mdx mice, the

mutation does not result in the same large fibrotic lesions seen in human DMD (Gillis

1999) and the mice appear to be able to regenerate their skeletal muscle (Grounds et al.

2005), yet certain muscles (such as the diaphragm) do, however, display some of the

features seen in human DMD patients (O'Brien and Kunkel 2001). In addition, the

serum of mdx mice has been reported to contain high levels of muscle-derived pyruvate

kinase and creatine kinase, which was also seen in DMD (Bulfield et al. 1984).

The age at which the mdx mice are selected for muscle necrosis studies is of major

importance. As discussed in Section 1.3.2.1, histological examination of muscle

sampled from mdx mice that are younger than 3 weeks of age bears little resemblance to

that associated with the dystrophic phenotype. At around day 21, however, there is an

acute onset of muscle damage in limb and paraspinal muscles (Coulton et al. 1988;

McGeachie et al. 1993; Grounds and Torrisi 2004; Shavlakadze et al. 2004). In these

mice, it has been reported that the percentage of muscle necrosis in the tibialis anterior

muscle increases to approximately 20-40% in 3 week old mdx mice (Shavlakadze et al.

2004). This percentage then decreases to around 6% at approximately 6 weeks of age as

result of muscle regeneration (McGeachie et al. 1993). For this reason, voluntary

exercise induces a significant increase in quadriceps muscle necrosis in mice (Archer et

al. 2006; Hodgetts et al. 2006; Radley and Grounds 2006). For this to occur, however,

mice from each treatment group are ideally required to run similar distance so that any

changes to the amount of myofibre necrosis measured in the quadriceps can be

compared to the treatment, thus eliminating the possibility that these differences are a

direct result of the varying distances covered.

In this study at least half of the mice failed to run and for those that did, the distances

recorded were minimal (Table 6.1). From these results, it is therefore not possible to


vivo in mdx mice

103

determine whether JNK inhibition by TAT-TIJIP would have a beneficial effect on

reducing myofibre necrosis in situations of muscle damage and wasting, as the levels of

myofibre necrosis seen in both the TAT-TIJIP treated, and more importantly the

untreated exercised mice (Figure 6.1), were relatively low and resembled normal levels

of myofibre necrosis observed at 6 weeks of age (McGeachie et al. 1993).

These results clearly demonstrate the need for further studies to be performed to

ascertain the therapeutic potential of inhibiting the JNK pathways by using TAT-TIJIP,

or other pharmacological inhibitors of JNK, in in vivo situations of myofibre necrosis.

Chapter Seven General Discussion

104

Chapter Seven

General Discussion

Overview

It is widely accepted the cytokine TNF promotes skeletal muscle atrophy and

contributes to myofibre necrosis, whereas IGF-1 has a key role in maintaining skeletal

muscle mass. The implication of cross talk between TNF and IGF-1 in skeletal muscle,

however, is not well defined (Grounds et al. 2008) and the effect of administering both

TNF and IGF-1 to skeletal muscle fibres has not yet been reported previously.

The present series of experiments comprising this research project were undertaken in

an attempt to determine the mechanisms of by which TNF induces atrophy in

differentiated muscles cells; specifically the involvement of TNF activated JNK and its

role in mediating cross-talk between IGF-1 and TNF. The initial experiments,

established that immortalised and primary mouse skeletal myotube tissue culture models

(IGF-1) induced myotube hypertrophy, and TNF induced myotube atrophy (Chapters 3

and 4). This was then followed by experiments designed to: (i) assess and

morphologically quantify the physiological effects of IGF-1, TNF and combined IGF-

1/TNF treatments (Chapter 4); (ii) confirm that TNF induces the phosphorylation of

JNK in the TNF induced skeletal muscle atrophy model developed using C2C12 cells

(Chapter 5); and (iii) use JNK inhibitors to decipher the role of JNK in mediating TNF

induced myotube atrophy, with inhibition of JNK activity confirmed by assessing c-Jun

phosphorylation (Chapter 5). Preliminary in vivo studies with JNK inhibitors were also

undertaken (Chapter 6).

7.1 Establishment of myotube culture models of IGF-1 induced hypertrophy and

TNF mediated atrophy

The present study used the C2C12 mouse skeletal muscle cell line that forms well

differentiated myotubes in tissue culture (Lloyd et al. 2004) and primary cultures of

muscles from FVB mice (to further validate the results from the C2C12 studies). The


105

studies confirmed the use of the (LONGTMR3IGF-I) analogue of IGF-1 to induce

significant hypertrophy in C2C12 myotubes, thus confirms previous studies (Florini et

al. 1996; Rommel et al. 1999; Rommel et al. 2001; Latres et al. 2005). Refinement of

the published protocols involved treating the myotubes at day 7, after the onset of

differentiation (as opposed to day 3), in order to use more mature myotubes. This

resulted in the establishment of an improved model of IGF-1 induced myotube

hypertrophy. The refined model uses C2C12 myotubes differentiated for 7 days, the

stage at which they are reported to be fully differentiated (Tolosa et al. 2005) and thus

more closely resemble the morphology of adult myofibres (Burattini et al. 2004).

The same 7 day C2C12 myotube model was then used to test the morphological effects

of 3 days of TNF treatment. A novel observation was statistically significant myotube

atrophy within 3 days after TNF (20 ng/ml) had been administered; this atrophic effect

of TNF alone has not previously been demonstrated. It is proposed that the more mature

status of the 7 day myotubes (beyond the steep growth phase) allowed the catabolic

effects of TNF to be manifested. The results using C2C12 myotubes were supported by

experiments using primary myotube cultures generated from young FVB mice, whereas

the same 3 day treatment with TNF (20 ng/ml) induced a statistically significant

decrease in mean myotube width.

Prior to the present research, there is only one report of C2C12 myotube atrophy when

TNF was administered in combination with IFN- γ (Dehoux et al. 2007). The

establishment of a novel tissue culture model of TNF induced myotube atrophy is a

powerful tool for future studies to investigate the molecular mechanism responsible for

this TNF mediated atrophy (without the complication of additional signalling pathways

activated by IFN- γ).

7.2 Combination treatments to investigate the effects of IGF-1 and TNF

Previously, the over-expression of IGF-1 in the skeletal muscles of transgenic

mdx/mIGF-1 mice (IGF:C2 mice) has been shown to significantly reduce myofibre

necrosis (Shavlakadze et al. 2004); a protective effect suggested as being the result of

increased protein synthesis in the muscles. Based on this hypothesis, the combination of

IGF-1 and TNF treatments were tested in C2C12 myotubes, and in muscle cell cultures

generated from transgenic IGF:C2 mice.


106

In the C2C12 myotube cultures, the simultaneous treatment of IGF-1 (10 ng/ml) and

TNF (20 ng/ml), or the addition of IGF-1 before/after TNF treatment, had no effect on

the widths of the myotubes. These finding were further supported by studies using

IGF:C2 myotubes, where the addition of TNF again had no effect on myotube widths.

The results suggest, therefore, that these cytokines cancel out the effect of each other,

and thus TNF prevented IGF-1 induced hypertrophy, and IGF-1 prevented TNF

mediated atrophy.

7.3 Involvement of JNK in TNF mediated myotube atrophy and use of JNK

inhibitors

There is now strong evidence that TNF can inhibit IGF-1 expression (Frost et al. 2003),

by abrogating IGF-1 receptor signalling through the inhibition of tyrosine

phosphorylation of IRS-1 (Grounds et al. 2008). Previous research supports that the

inhibitory effect of TNF on IGF-1 is mediated by JNK (Broussard et al. 2003; Strle et

al. 2006). Detection of phosphorylated JNK, within 10 minutes of TNF treatment of

C2C12 myotubes, confirmed that TNF rapidly activates JNK (see Section 5.1.1). This is

a new contribution to the understanding of this process which has not been previously

published. To date, detection of TNF induced phosphorylated JNK has only been

reported in proliferating C2C12 myoblasts (Strle et al. 2006; Huang et al. 2007). Other

complementary studies in the Grounds laboratory using the same C2C12 7 day myotube

model, show that transcription of the atrogenes Atrogin-1 (MAFbx) and MuRF1 is

upregulated with 2-4 hours of TNF treatment, thus confirming the rapid upregulation of

genes responsible for the catabolic effects of TNF on myotubes (Chinzou 2009).

To determine if JNK activation played a critical role in TNF mediated atrophy, JNK

inhibitors were assessed for toxicity, and one (TAT-TIJIP) selected for testing in the

C2C12 and primary FVB myotube cultures (Chapter 5). Three JNK inhibitors were

evaluated; two that were ATP-competative (SP600125 and AS601245) and one that was

ATP-non-competitive (TAT-TIJIP). Both (SP600125 and AS601245) appeared toxic

when administered to day 7 C2C12 myotubes daily for 3 days whereas TAT-TIJIP was

not. The effect of TAT-TIJIP on the widths of the myotubes was therefore quantified in

the absence and presence of TNF (along with TAT controls). A striking effect was that

the inhibition of JNK activity by TAT-TIJIP prevented TNF mediated atrophy in all


107

three myotube models; e.g. with C2C12 and primary myotube cultures generated from

FVB and IGF:C2 mice.

A somewhat unexpected finding was that the combined treatment of TAT-TIJIP and

TNF resulted in hypertrophy in both C2C12 and primary FVB and IGF:C2 myotubes. It

was instead hypothesised that inhibition of TNF/JNK mediated atrophy would result in

myotube widths resembling those of the untreated cultures, however, TAT-TIJIP

appears to be activating pathways involved in myotube hypertrophy. This raises many

interesting questions regarding the signalling mechanism responsible for this

hypertrophy, but resolution of this lies beyond the scope of the present research project.

However, the change in phosphorylation and activation state of IGF-1-mediated

signalling, that includes the IRS-1/PI3K/AKT/mTOR pathway as well as key regulators

of protein synthesis (such as p70S6K and 4EBP-1 - Rommel et al. 2001; Latres et al.

2005) in response to TNF + TAT-TIJIP, are of significant interest for future studies.

Although the present study could not optimise an assay to detect activated IRS-1 in

C2C12 cells (probably due to low levels of such signalling in the cell line used), an

alternative indirect approach was used to confirm the inhibition of JNK activity by

TAT-TIJIP based on phosphorylation and nuclear translocation of c-Jun; a downstream

target of phosphorylated JNK. Immunohistochemical analyses showed nuclear

localisation and phosphorylation of c-Jun in TNF treated myotubes, but not in cultures

pre-treated with TAT-TIJIP before TNF treatment, nor in the untreated control

myotubes. It has been shown TNF activates Atrogin-1 when administered to

differentiated C2C12 myotubes (Li et al. 2005) and, as discussed above, the Grounds

laboratory has also demonstrated rapid TNF mediated upregulation of both Atrogin 1

(MaFbx) and MuRF1 in C2C12 myotubes (unpublished data). Based on these findings,

the specific effects of TAT-TIJIP on transcription of these atrogens could be determined

in future experiments

These combined data describe a new cell culture model for TNF mediated myotube

atrophy, with the results presented in this thesis strongly supporting a role for JNK in

TNF mediated muscle atrophy. The results identify new aspects of cross-talk in

signalling between the TNF and IGF-1 pathways in myotubes and provides a strong


108

basis for the use of JNK inhibitors for future pre-clinical in vivo studies in mouse

models of muscle disorders

7.1.4 In vivo studies: preliminary findings

The inconclusive results from the in vivo experiments in this thesis highlighted the

importance of refining many parameters, (e.g. dosage, voluntary exercise protocol)

before in vivo testing can be thoroughly investigated

The initial in vivo study was encouraging, because there were no apparent adverse

effects after treating mice daily for 3 days with 10 mg/kg TAT-TIJIP. There is little

information available on the ideal dosage and route of delivery of such JNK inhibitors

in animals and no toxicology studies performed to assess any potential toxic side effects

when administered in vivo. Therefore, the experimental methodology was arbitrarily

based on a study, which tested in vivo, an inhibitor similar to TAT-TIJIP (Kaneto et al.

2004). Due to time limitations, the issues of toxicity, optimal dosage and ideal delivery

route could not be addressed and this needs to be taken up future research. The potential

of JNK inhibitors to reduce muscle wasting in vivo is strongly supported by the present

tissue culture studies of myotubes, and is an area of considerable interest for future

research into the possible use of such inhibitors as therapeutic interventions for muscle

wasting disorders, such as DMD and cachexia.

Based on the findings presenting in this thesis, the promising results of TAT-TIJIP in

preventing TNF-induced myotube atrophy demonstrates the potential of this JNK

inhibitor to be of therapeutic benefit for muscle wasting disorders, such as cachexia and

DMD. Through future analysis of cell signalling pathways, and eventual in vivo testing,

the specificity and possibility of using TAT-TIJIP (and other JNK inhibitors) as

therapeutic agents will begin to be understood with the optimum result being

implemented as a potential therapy for muscle wasting disorders.

References

109

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