devel op men t of novel inte rventions to enhance muscle...

D evel op men t of Novel Inte rventionsto Enhance Muscle Function

b y

Pat ricio Vicente Sepulveda SalinasBApplSc ( Hons.)

Sub mitted in fulfilment of the requirements for the degree of

Doctor of Philosophy

Deakin University

O c tobe r, 2012

sfol

Retracted Stamp

iv

ABSTRACT

The aim of this research was to test the effect of follistatin (Fst) and or Peroxisome

Proliferator co-activator 1 alpha (PGC-1 ) overexpression with recombinant adeno

associated viral vectors (rAAV) in modulating muscle function. The introduction

(chapter I) was written to provide background information on skeletal muscle

physiology. Chapter I focused on skeletal muscle function, the regulation of its mass

and metabolism. Chapter II described material and methods and it was followed by

experimental chapters.

The first aim of this thesis was to test the anti-atrophy effect of rAAV-Fst. The results

in chapter III showed that rAAV-Fst increases muscle mass and protein synthesis.

This response was capable of preventing non-degenerative muscle wasting (nerve

and tendon injury) in C57/BL6 mice when delivered 2 weeks prior to injury. However,

muscles treated with rAAV-Fst failed to respond when the vector was delivered at

the time of injury. The results suggested an intact nerve and mechanical load are

required for Fst-mediated muscle growth.

The second aim of this thesis was to test the metabolic effect of Fst. In chapter IV,

the results showed that 2 week treatment with rAAV-Fst led to decreased glucose

uptake and fatty acid metabolism. In parallel, rAAV-Fst produced an increase in the

proportion of diacylglicerol (DAG) esterification and accumulation. Despite this,

rAAV-Fst treated muscles had increased sensitivity to insulin-meditated glucose

uptake and decreased response to contraction-induced glucose uptake. These

suggested a positive role for rAAV-Fst induced growth in the regulation of glucose

metabolism.

v

The third aim of this thesis was to test the effects of rAAV- PGC-1 , rAAV-Fst and

their combination. The results in chapter V showed that acute (2 weeks) post-natal

delivery of rAAV-PGC-1 offered no protection against denervation. Chronic

treatment (8week) with rAAV-PGC-1 improved glucose metabolism, reduced peak

force and increased resistance to fatigue. On the other hand, chronic treatment with

rAAV-Fst produced larger and stronger muscles. Co-delivery of rAAV-PGC-1 and

rAAV-Fst resulted in very large muscles, however they produced less force and had

decreased resistance to fatigue. These results suggested that PGC-1 and Fst exert

some adaptations that are mutually exclusive.

A general discussion and conclusion (chapter VI) was written to highlight the most

important findings of this thesis and debate about the importance and implications of

this research.

vi

PREFACE

This thesis comprises my own work, except as indicated below:

The transfection of packaging/transgene plasmids and the purification of the

resulting rAAV vectors for the use in this study were made by Dr. Paul

Gregorevic or Dr. Hongwei Qian (Baker IDI Heart and Diabetes Institute, VIC,

Australia).

Initial denervation and tenotomy surgeries were performed by Dr. Paul

Gregorevic while he was instructing me on how to perform these procedures.

Western Blots in Chapter two were prepared by Dr. Catherine Winbanks

(Baker IDI Heart and Diabetes Institute, VIC, Australia).

Some glycogen measures in Chapter 4 were partially performed by Dr. Robert

Lee-Young (Baker IDI Heart and Diabetes Institute, VIC, Australia).

Dr. Robert Lee-Young and Christine Yang (Baker IDI Heart and Diabetes

Institute, VIC, Australia) helped with cannulation surgeries to obtain results in

Chapter 4 and 5.

Joanne Bab (Baker IDI Heart and Diabetes Institute) helped with the

extractions of lipids and Jacqui Weir (Metabolomics Laboratory of the Baker

IDI Heart and Diabetes Research Institute) provided guidance for the analysis

of mass spec data was receive

ANOVA statistical analysis was performed by Dr. Séverine Lamon (Deakin

University, VIC, Australia)

vii

ACKNOWLEDGEMENTS

Attaining the degree of Doctor of Philosophy is a great achievement that

encompasses the support and effort from many, and thus I wish to recognize the

following:

To my scientific supervisor Dr. Paul Gregorevic, first of all I like to thank you

for the opportunity to access your laboratory and scientific training. I value

your intellectual input and support throughout my PhD. For these, I thank you

for your contribution towards my professional development.

To my principal supervisor Dr. Aaron Russell, I really do not know how to

begin to thank you… but I will try. You are not just a bright scientist but also a

great person and friend. If it wasn’t for our relationship, this PhD would have

not occurred. Thank you for looking after me during the last couple of years. I

consider myself very lucky of having encountering you on my path.

To my colleagues and collaborators Dr. Clinton Bruce and Dr. Robert Lee-

Young. Guys, without the two of you I would not be writing this right now.

Thank you for inspiring, supporting and lending me a hand in the lab. It has

been a lot of fun to work hard next to you. I appreciate the input and direction

that you gave to my research.

To Dr. Catherine Winbanks, we shared the lab and many coffees over 3

years. I really enjoyed working with you. I admire your integrity and

perseverance and I believe your work will get you very far. You are truly an

example for early career scientists. Thank you for your support.

To Dr. Séverine Lamon, for efficient and accurate assistance with statistical

methodology and analysis of data.

viii

I would like to thank the friends of the Baker IDI, in particular, Jan and Robert

Lyng, for your support to the ‘Bright Sparks program’ that helped me

throughout my PhD.

I would like to thank Professor Mark Febbraio, for opening the doors of your

laboratory and providing the support for my doctoral investigation.

To the muscle lab, a family of people that supported my PhD journey. Thank

you for the help and advice.

To Marita, Craig, Erin and Renea, thanks for good (and amusing) times.

To my mum, Patricia, thank you for believing on me and supporting me

through times of hardship.

To my dad, Patricio. I will be forever grateful of your support, advice,

understanding and encouragement, no matter what it is that I am doing.

To my brother, Pablo Sepulveda for always being there for me.

Last, but most definitely not least, I would like to thank my wife Maria

Smetanina, for filling my life with love and helping me to move forward. Maria

you are my biggest inspiration! I appreciate all the sacrifices you have done

for me, now it will be my turn to take care of you through the journey of life.

ix

TABLE OF CONTENTS

ABSTRACT .............................................................................................. iv

PREFACE ................................................................................................ vi

ACKNOWLEDGEMENTS ....................................................................... vii

TABLE OF CONTENTS .......................................................................... ix

TABLE OF ABBREVIATIONS .............................................................. xiv

CHAPTER I – INTRODUCTION ............................................................. 24

General introduction ........................................................................................... 25

The health of skeletal muscle plays a key role in our lives ................................ 25

Developing new approaches to improve skeletal muscle health is imperative ... 25

Using gene therapy to enhance skeletal muscle function .................................. 26

Introduction ......................................................................................................... 28

Fundamental muscle biology ............................................................................. 28

Mechanics of muscle contraction ....................................................................... 33

Skeletal muscle health ....................................................................................... 35

Regulation of post-natal skeletal muscle growth ................................................ 36

Skeletal muscle metabolism .............................................................................. 71

Gene therapy with rAAV .................................................................................... 75

Aims and hypothesis .......................................................................................... 81

x

Aims ................................................................................................................... 81

Hypothesis ......................................................................................................... 81

Implications of this work ..................................................................................... 82

References........................................................................................................... 83

CHAPTER II – MATERIALS AND METHODS ..................................... 117

General reagents ............................................................................................... 118

Tissue culture .................................................................................................... 118

In vitro transfection of plasmids ...................................................................... 118

Standard cloning techniques ........................................................................... 119

Viral Vector production .................................................................................... 119

Preparation of rAAV expression plasmids ....................................................... 119

Generation of viral vectors ............................................................................... 120

Mouse handling and interventions .................................................................. 122

Intramuscular injection of rAAV........................................................................ 123

Muscle Denervation ......................................................................................... 123

Tenotomy ......................................................................................................... 124

End-point tissue collection ............................................................................... 124

Muscle contraction ........................................................................................... 125

Insertion of catheter into the jugular vein ......................................................... 126

Body composition ............................................................................................ 127

Radioactive Tracer Experiments ..................................................................... 127

xi

Ex vivo protein synthesis assay ....................................................................... 127

Ex vivo protein degradation assay ................................................................... 128

Ex vivo glucose metabolism experiments ........................................................ 129

In vivo insulin-stimulated glucose uptake experiments .................................... 129

Determination of intramuscular glycogen content ............................................ 130

Ex vivo fatty acid metabolism experiments ...................................................... 131

Histology............................................................................................................ 132

Preparation of samples .................................................................................... 132

Haematoxylin and eosin stain .......................................................................... 133

Immunofluorescent labelling ............................................................................ 133

DNA and RNA analysis ..................................................................................... 134

Western Blotting for protein abundance......................................................... 135

Biochemical analysis ........................................................................................ 136

-hydroxyacyl-CoA dehydrogenase ( -HAD) activity assay ............................ 136

Citrate synthase (CS) activity assay ................................................................ 136

Lipidomics analysis .......................................................................................... 137

Statistical analysis ............................................................................................ 139

References......................................................................................................... 140

CHAPTER III – GROWTH AND NON-DEGENERATIVE MUSCLE

WASTING .............................................................................................. 142

Summary............................................................................................................ 143

xii

Introduction ....................................................................................................... 144

Aims/Hypothesis .............................................................................................. 146

Significance of proposed work/outcomes ........................................................ 147

Results ............................................................................................................... 148

Discussion ......................................................................................................... 160

References......................................................................................................... 165

CHAPTER IV – MUSCLE GROWTH AND ENERGY METABOLISM . 170

Summary............................................................................................................ 171

Introduction ....................................................................................................... 172

Hypothesis/Aims .............................................................................................. 175

Significance of this work .................................................................................. 175

Results ............................................................................................................... 176

Discussion ......................................................................................................... 187

References......................................................................................................... 190

CHAPTER V – ENGINEERING A FATIGUE RESISTANT AND

STRONG MUSCLE ............................................................................... 193

Summary............................................................................................................ 194

Introduction ....................................................................................................... 195

Aims/Hypothesis .............................................................................................. 196

Significance of this work .................................................................................. 197

Results ............................................................................................................... 198

xiii

Discussion ......................................................................................................... 209

References......................................................................................................... 212

CHAPTER VI – DISCUSSION AND CONCLUSSION ......................... 217

General discussion and future directions ...................................................... 218

Conclusion ........................................................................................................ 223

References......................................................................................................... 224

CHAPTER VII – APPENDIX ................................................................. 225

Appendix 1. Ethics 1828 ................................................................................... 226

Appendix 2. Ethics 2265 ................................................................................... 242

Appendix 3. Ethics 2039 ................................................................................... 258

Appendix 4. Troubleshooting of rAAV expression in vitro ........................... 273

xiv

TABLE OF ABBREVIATIONS

4E-BP1 PHAS-1

5-Aminoimidazole-4-carboxamide ribotide AICAR

Acid Soluble portion of metabolism ASM

Activin receptor II A ActRIIA

Activin receptor II B ActRIIB

Adeno Virus AdV

Adenosine diphosphate ADP

adenosine monophosphate AMP

Adenosine triphosphate ATP

adenosine monophosphate kinase AMPK

Akt isoform 1 Akt1

Akt residue Serine 473 Ser473

Akt residue Threonine 309 Thr309

Ampicillin amp

Amyotrophic Lateral Sclerosis ALS

apoptotic repressor with caspase recruiting domain ARC

Atrogin-1 and MuRF1 atrogenes

xv

B cell lymphoma protein 2 Bcl-2

Bcl-2 associated X protein BAX

Beta oxidation B-oxidation

Beta(3)-Hydroxycyl-Coa Dehydrogenase -HAD

bismonoacylglycerolphosphate BMP

BMP Activin membrane-bound inhibitor Bambi

Bone Morphogenic Protein BMP

Bovine Serum Albumin BSA

C57 Black 6 mouse C57Bl/6

Calcium ions Ca+2

cancer-induced atrophy cachexia

capsid cap

Carbon dioxide CO2

Carnitine palmitoyltransferase 1 CPT 1

Central nervous system CNS

ceramide Cer

Chronic obstructive pulmonary disease COPD

complementary DNA cDNA

xvi

Creb binding protein CBP

cycloheximide CHX

cytokine kinase protein 38 p38MAPK

cytomegalovirus CMV

Citrate Synthase CS

Delta

Denervation Den

Deoxyribonucleic acid DNA

diacylglycerol DAG

DMD mice mdx

Dulbecco’s Modified Eagle's Medium DMEM

Duchenne Muscular Dystrophy DMD

Dystrophin Glycoprotein Complex DGC

E1A binding protein p300 p300

Elongation Initiation Factors eIFs

Estrogen related Receptor alpha ERR

Extensor Digiturium Longus EDL

extracellular matrix ECM

xvii

fatty acid FA

FKHR Foxo3a

flavoprotein apoptosis-inducing factor AIF

foetal bovine serum FBS

Follistatin Fst

Fork Head Transcription Factor 1 FKHR

Forkhead box O FoxO

Free Fatty Acids FFAs

FST long 315aa Fst315

FST short 288aa Fst288

Glucocorticoid Receptor GR

Glucose transporter 1 GluT-1

Glucose transporters 4 GluT-4

Glycogen Synthase kinase 3 beta GSK-3

Growth associated serum protein 1 Gasp-1

Haematoxylin and eosin H&E

Hanks Balanced Salt Solution HBSS

Hertz Hz

xviii

Horse Serum HS

Human Embrionary kidney cells 293 D HEK293D

Human fibrosarcoma cells HT1080

human Placental alcohol phosphatase hPLAP

I-Kappa Beta IkB

IkB kinase Beta IkBk

Inhibitor of NF-k IKK

Inhibitor of NF-k 2 IKK2

initiation factor 4E binding protein 1 4E-BP1

Insulin Growth Factor 1 IGF-1

Insulin receptor substrate 1 IRS-1

Inverted Terminal Repeats ITRs

Knock out KO

Kreb's-Henseleit buffer KHB

lysophosphatidylcholine LPC

MAFBx Atrogin-1

mammalian target of rapamycin mTOR

micro Curie Ci

xix

millilitre mL

minute(s) min

molar M

Mouse myoblast C2C12

Multiple Reaction Monitors MRM

muscle RING-finger protein-1 MuRF1

muscle specific f-box protein MAFBx

Myocyte Enhancing Factor 2 MEF2

Myogenic regulator factor D MyoD

myosin heavy chain MyHC

Myosin light chain MyLC

National Health and Medical Research Council NHMRC

National Institute of Health NIH

Neuro-muscular junction2 NMJ

Neutral Loss NL

Nitric oxide synthase NOS

Nitrogen N2

nuclear factor kappa beta NF-k

xx

Nuclear Respiratory Factor 1 NRF1

Nuclear Respiratory Factor 4 NRF4

Optimum muscle length L0

P70 S6 kinase P70S6K

pAAV-Follistatin-288 pAAV-FST

penicillin and streptomycin P/S

peroxisome proliferator activator receptor PPAR

Peroxisome Proliferator-activated receptor gamma

coactivator 1 alpha PGC-1

phosphatidylethanolamine PE

phospharidylinositol PI

Phosphate Buffered Saline PBS

phosphatidylcholine PC

phosphatidylethanolamine PE

phosphatidylglycerol PG

phosphatidylserine PS

Phosphoinoisitide 3-kinase PI3K

phosphorylated Akt p-Akt

xxi

phosphorylated mTOR p-mTOR

phosphorylated S6RP pS6RP

plasmid type-6 pseudotype capsid gene pDGM6

PPAR delta PPAR

rAAV plasmids pAAV

rAAV serotype 2 rAAV2

rAAV serotype 6 rAAV6

rat myoblast L6

recombinant adeno-associated viral vector rAAV

recombinant adeno-associated viral vectors rAAV

replication rep

S6 Ribosomal protein S6RP

Smad isoform 1 Smad1






xxii

Small mother of decapenthaplegic Smad

sodium dodecyl sulphate SDS

Sodium Hydroxide NaOH

sphingomyelin SM

sphingosine Sph

Spinal Muscular Atrophy SMA

Standard Deviation SD

Standard Error of the Mean SEM

Super Optimal Broth SOC

TBS containing 0.1% Tween-20 TBST

Tenotomy Ten

Thymona viral oncogene or protein kinase B Akt

Tibialis Anterior TA

TNF-like weak inducer of apoptosis TWEAK

Transcription Factor A Tfam

Transforming Growth Factor Beta TGF-

Transgenic Tg

transverse tubule T-tubule

xxiii

triacylglycerol TAG

trihexosylceramide THC

Tris-buffered saline TBS

Tumour Necrosis Factor TNF

tumour protein 53 p53

Ub-activating enzyme E1

Ub-conjugating carriers E2s

ubiquitin proteasome pathway UPP

Ub-protein ligase E3

Unit(s) U

Ying-Yang 1 YY1

glucose uptake Rg’

glucose clearance Kg’

tritium labelled 2-deoxy-D-glucose 3H-2DG

distilled water d-H2O

Analysis of variance ANOVA

24

CHAPTER I – INTRODUCTION


25

General introduction

The health of skeletal muscle plays a key role in our lives

Skeletal muscle, without doubt the largest organ of the human body, plays a

fundamental role in human health and wellbeing. Skeletal muscle constantly

adapts its mass and metabolism to accommodate demands in response to

health status. In adults, interventions promoting muscle mass increase

(hypertrophy) have positive health consequences such as delaying the onset

and reducing severity of disease (1). In contrast, skeletal muscle loss

(atrophy) aggravates the seriousness of health affecting disorders (2-4). In

addition, atrophy decreases the quality of life in the elderly and reduces their

ability to respond and/or recover from disease (5). The loss of muscle mass

has been associated with unfavourable changes in the metabolism of

proteins, carbohydrates and lipids (6, 7). Atrophy is also believed to contribute

to the burden of metabolic disorders (8-11). These are the reason why the

direct and indirect economic costs associated to atrophy are believed to add

as much as half of the Australian yearly health expenditure (12).

Unfortunately, there is no treatment against atrophy. Hence, new therapeutic

approaches to tackle muscle atrophy could help improving many lives and

save billions of dollars.

Developing new approaches to improve skeletal muscle health is

imperative

It has been long known that the ability to ambulate and exercise is

fundamental to human life health. The evidence pointing to exercise as a

potential therapeutic intervention to prevent or ameliorate cardiovascular


26

conditions (obesity, heart disease), cancer, osteoporosis and atrophy (13)

keeps growing. However, one of the major drawbacks of modern society is

the lack of exercise. Sedentary behaviours are on the rise and contributing to

the alarming increase in (mostly preventable) metabolic disorders such as

insulin resistance, obesity and diabetes. Adding to this problem is that as a

result of today’s improved health care, there is an increase in the elderly

population as well as the number of patients critically and chronically ill

confined to hospital beds (14). Unfortunately, the elderly, ill and sedentary

people cannot benefit from regular exercise. Hence, there is a need to better

understand the biology of muscle and create interventions to improve it.

Fortunately, there is hope of new avenues of therapeutic developments such

as novel ways to deliver transgenes that allow us to manipulate the

physiology of skeletal muscle in ways never seen before (15-18).

Using gene therapy to enhance skeletal muscle function

Identification of novel methods to mimic or potentiate the effects of exercise is

a long standing, albeit elusive medical goal. Lately, new tools for controlling

genes in non-transgenic animals exist via administrating recombinant adeno-

associated viral vectors (rAAV). These vectors provide the means to explore

the physiological significance of controlling gene expression in post-natal

muscle. In the context of exercise-related adaptation of muscle, rAAV rise

high above the rest by their ability to allow tissue specific, long lasting and

non-pathogenic transgene expression. Two target proteins that could benefit

from their delivery with viral vectors are Follistatin (Fst) and Peroxisome

Proliferator-activated receptor gamma co-activator 1 alpha (PGC-1 ). A body

of evidence points to Fst as a potential positive regulator of muscle mass in


27

health and disease (19-21). PGC-1 , on the other hand, is a key protein

involved in modulating skeletal muscle’s oxidative metabolism (22-24).

The following introductory chapter presents a review on the relevant literature

about the origin of skeletal muscle and the key regulatory pathways involved

in skeletal muscle growth and metabolism. In particular, the introduction

highlights how Fst and or PGC-1 in the context of atrophy and metabolism.


28

Introduction

Fundamental muscle biology

Musculature formation (myogenesis) is a sophisticated process beginning as

early as the embryo has three defined layers: the outer, inner and middle

layer (or ecto-, endo- and mesoderm) [reviewed in (25-28)]. Myogenesis

occurs within the mesoderm’s compartment called myotome (for muscle) (29).

In the myotome, myogenic progenitor cells (myoblasts) align and fuse into

multinuclear mature muscle cells (myotubes) as a part of their commitment to

form myofibres, the building blocks of muscle (30). Once adult skeletal muscle

is formed, it still retains a subpopulation of dormant myoblasts (satellite cells)

which become active to support muscle repair, growth and maintenance (27,

28, 31-33).

A key structural characteristic of skeletal muscle is that myofibres are made of

longitudinally arranged sacomeres containing mechanical filaments which

appear like “light” and “dark” bands when their protein/filament arrangement is

observed under high magnification (Figure 1A). Light bands or I-bands

comprise parallel arrays of thin filaments containing Actin (34) (Figure 1B).

Actin, the most abundant protein in skeletal muscle (35), spans its filaments

into the dark bands or A-bands where the myosin-containing thick filaments

are found. Actin and myosin interact to create tension (also known as muscle

contraction) via a process known as cross-bridge action (36) (Figure 1C).

Generation of tension and cross-bridge action are discussed in section

‘Mechanics of muscle contraction’ (page 32). Muscle contraction is controlled

by influx of Ca2+ and depends on tropomyosin and troponin to generate force


29

(37). Many accessory proteins, including an additional filament system made

of Titin or nebulin, span through the sarcomere. For example, the boundary of

the sarcomere (Z-discs) contain a capping protein, CapZ, and serves to

anchor the actin, titin and nebulin filaments (38) and is implicated in

mechanosensation and signalling (39). Also found in the muscle cells are

invaginations of its membrane (also called sarcolemma) corresponding to

perpendicularly penetrating transverse tubules (T-tubule) (Figure 2). T-tubules

unify the depolarisation of the muscle membrane allowing a synchronised

contraction.

The periphery of muscle cells is characterized by the presence of multiple-

nuclei (Figure 2). These nuclei result from recruitment and fusion of single-

nuclei muscle cells (myoblasts) early in development. Also in the periphery it

is possible to observe that the sarcolemma is in contact with controlling motor-

nerve (Figure 2). Each of these sites is known as a neuromuscular junction

(NMJ). Motor-nerves originate at motorneurons, a part of the specialised

brain-to-muscle circuit (cell body, neural processes and NMJ) directing the

electrical control of skeletal muscle. One motor unit can innervate up to 1900

muscle fibres (such as the gastrocnemius). During development, motor units

reach immature muscle fibres with multiple axons. Once these myogenic

fibres are contacted they start to differentiate into primary and secondary

myotubes [reviewed in (40)]. Pre-natal muscle fibres undergo myogenesis

simultaneously with the establishment of multiple motorneurons. After birth,

the immature muscle undergoes a process of motorneuron elimination (41),

which correlates with a progressive change from embryonic to mature myosin

heavy chain (MyHC) composition (42). Motorneuron elimination leads to a


30

single axon attached per fibre in the adult muscle (43). Big axons derived from

big motorneurons recruit the less active (fast) fibres, while the opposite is the

case of very active (slow) fibres (44).

Figure 1. A myofiber containing dark and light striations and external nuclei (A). Each myofiber is composed of aligned myofibrils on which the light bands are rich in actin and the dark bands correspond to enrichment of myosin overlapping with Actin (B). Actin and myosin are organised in a structure called sarcomere (C) which is the basic contractile unit of the muscle (45).


31

Figure 2 shows a myofiber contacted by a motorneuron via axons that contact the sarcolemmal NMJ. A myofiber contacts a single axon at multiple sites allowing a coordinated contraction among all the myofibrils at the same time (45).

There are many specialized structures in the muscle cells. Microtubules are

the building blocks of an integration network. Microtubules link the

sarcolemma, Golgi complex and nuclei {Kano, 1991 #1442}. Costameres are

another structure found affixed to the sarcolemma. Costameres contribute to

mechanosignal transduction and provide an anchoring site for the

extracellular matrix (ECM) proteins. The ECM is vital in preserving the

structural integrity of the sarcolemma during muscle contraction.

There are other important networks that support the correct function of

skeletal muscle. The first, focal-adhesions, anchor the ECM to actin proteins


32

via integrins. Integrins are involved in myofibril assembly and

mechanosensing [reviewed (46)]. The second is known as the dystroglycan

complex (DCG). The DCG hosts dystrophin in its core and binds the

cytoskeleton to the ECM [for details see (47-49)]. Genetic disorders affecting

the DCG are responsible for the most common class of genetic myopathy,

Duchenne Muscular Dystrophy (DMD). The third is the spectrin/ankyrin

complex which is situated in the Z-line and M-lines (50-54).

Skeletal muscle force is generated from the interaction between myosin and

actin, two structural proteins with a contractile function. There are two types of

Myosin, myosin heavy chain (MyHC) and myosin light chain (MyLC). MyHC

has two clear portions, the head and the rod (55). The head interacts with

actin via an active process requiring adenosine triphosphate (ATP) (56).

There are different myosin isoforms that fall under two main categories, fast or

slow, depending on the speed they act (57, 58). MyHC type IIa (MyHCIIa),

type IIx (MyHCIIx) (human) and type IIa (MyHCIIa) (mouse) are predominantly

fast isoforms expressed in skeletal muscle while MyHC type I (MyHCI) is

predominant slow (59). Other classes of myosin are also found in the heart,

extra ocular muscles and during development, however their discussion goes

beyond the scope of this review [reviewed in (60)]. Muscle contractions not

only generate the force required for movement, but also they are capable of

modify the properties of skeletal muscle (61-64). The following section

describes the mechanics of muscle force as a preamble to the insights of

muscle health and its post-natal regulation.


33

Mechanics of muscle contraction

A voluntary muscle contraction is the end result of a chain of events. Electric

waves generated from the brain travel through the central nervous system

(CNS), the motor neurons and axons. Once an electric pulse reaches the

sarcolemma, it leads to influx of ions through calcium-dependent channels

(65). Influx of calcium ions (Ca2+) causes the release of acetylcholine into the

extracellular space (between the motor neuron terminal and the motor end

plate). Ca2+ binds to nicotinic acetylcholine receptors and opens the

sodium/potassium channels. This process causes the membrane to become

depolarised, triggering an action potential. Spreading of the action potential

occurs through the network of T-tubules, depolarising the inner portion of the

muscle fibre. Depolarisation activates voltage-dependent calcium channels

(dihydropyridine receptors) which are in close proximity to calcium-release

channels (ryanodine receptors) in the adjacent sarcoplasmic reticulum.

Activated voltage-gated calcium channels physically interact with calcium-

release channels causing the sarcoplasmic reticulum to release calcium. The

increased cytosolic concentration of calcium leads to its binding to troponin C

which modulates tropomyosin (66). This causes an allosteric change in

troponin protein T which in turns allows tropomyosin to move, unblocking the

myosin binding sites (67). Myosin binds to the newly uncovered binding sites

on the actin filament to facilitate the power stroke (68). As a result, ADP and

inorganic phosphate are released. This will pull the Z-bands towards each

other, thus shortening the sarcomere (muscle tension). In order to relax, ATP

binds to myosin, allowing it to release actin. Myosin then hydrolyses the ATP

and uses it to return to an initial pre-contraction state and active re-synthesis


34

of ATP takes place. While the above steps are occurring, special transporters

actively pump calcium back into the sarcoplasmic reticulum to maintain its

ionic concentration in the sarcoplasm. Thus, the tropomyosin-troponin

complex again covers the binding sites and the sarcomere returns to initial

state. The maximal tension produced during muscle contraction correlates

with the number and size of fibres recruited, thus it is generally believed that

muscle mass is proportional to force generating capacity. Therefore, it is easy

to understand why maintenance of muscle and function is important for overall

wellbeing.


35

Skeletal muscle health

Skeletal muscle, nearly half of a human's body mass, is a unique tissue that

significantly contributes to an individual's locomotion, metabolism and overall

quality of life. The main determinants of skeletal muscle health are its mass

and metabolism (handling of carbohydrates, lipids and proteins).

Changes in skeletal muscle mass and metabolism are closely interconnected.

In healthy individuals, developmental growth and exercise are the main

factors affecting energy requirements (69). During childhood and particularly

in teenagers, circulating growth factors produce an incremental gain in muscle

mass and energy requirements/utilization. However, once developmental

maturity peaks and circulating growth factors decline, maintenance of muscle

function relies on regular exercise and diet. Although the concept is very

simple: exercise and stay healthy, a vast percentage of the world’s population

suffers from the deleterious effects of a sedentary lifestyle. Often, elderly and

chronically ill people also suffer the consequences from lack of exercise. In

combination, these populations represent a burden for the public and private

medical systems. Thus, there is a clear need for treatments with the potential

to enhance muscle function in people that are not doing or can’t do enough

physical activity.


36

Regulation of post-natal skeletal muscle growth

Muscle mass gains occur during developmental growth, in response to growth

factors (70, 71), dietary changes and increased exercise load (72). Upon

repeated load, skeletal muscle increases its size, mass and strength.

Increasing skeletal mass involves some or all the following processes:

addition of myogenic precursor cells (satellite cells and other precursor cells)

(73-77), increased number of fibres (hyperplasia) (78, 79) and/or muscle fibre

hypertrophy (80) .

The inclusion of satellite cells to the existing myotubes occurs mainly during

developmental growth and post-natal muscle repair. Upon activation, satellite

cells proliferate, differentiate and fuse with existing myotubes (77).

Histologically, newly fused satellite cells appear at the centre of cross

sectional muscle fibres. As myotubes mature, satellite cells migrate to the

periphery of the muscle cell [reviewed in (81)] (Figure 3).

Figure 3 shows an schematic representation of the steps required for satellite cells to become part of myofibres [Figure published by (81)].

Hyperplasia is a process that rarely occurs in adult muscle, but it is rather

confined to pre-natal development. However, there are reports of post-natal

hyperplasia. It would appears that this takes place when an existing myofiber


37

invagination branches and splits (82) or during acute phases of growth in

rodents (83).

The most prominent form of post-natal muscle growth is skeletal muscle

hypertrophy. Muscle hypertrophy is controlled by two contrasting processes,

protein synthesis (anabolism) and degradation (catabolism) (84) [reviewed in

(85-91)]. These processes react in response to natural and synthetic

interventions. The lessons learnt from physiological growth has led to the

development of many synthetic approaches for the regulation of muscle mass

(92, 93). These interventions have been designed to mimic, amplify or block

subset signalling pathways implicated in muscle growth/wasting (87, 94, 95)

and may in turn impact in hundreds, if not thousands, of muscle remodelling

genes/gene regulators (96-103). This thesis focused in the Thymona viral

oncogene (Akt)/ mammalian target of rapamycin (mTOR) (104) and the

Transforming Growth Factor Beta (TGF- ) pathways (105, 106) for their

protagonist role in the regulation of mass. In addition, this thesis discussed

the role of Peroxisome Proliferator co-activator 1 alpha (PGC-1 ) as a key

regulator of muscle metabolism.

The Akt/mTOR pathway

Activation of Akt/mTOR is one of the key events involved in exercise mediated

muscle growth (107-110). An increase in Akt phosphorylation [serine residue

473 (Ser473) threonine 309 (Thr309)] can command the up-regulation of

protein synthesis and down-regulation of protein degradation. The latter

occurs via phosphorylation (and inhibition) of Fork Head Transcription Factor

1 (FKHR) also known as forkhead box O (FoxO) (111-116). Akt controls

protein synthesis, primarily, via mTOR (104, 117) through inhibition of


38

glycogen synthase kinase 3 beta (GSK-3 ) (112). Phosphorylation of GSK-3

removes its inhibition of elongation initiation factors (eIFs) that influence

translation initiation resulting in increased protein synthesis in skeletal muscle

(118). mTOR also acts through phosphorylation of protein 70 S6 kinase

(p70S6K) which in turn phosphorylates and represses the catabolic activity of

the eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) also

known as PHAS-1 (87, 88) (Figure 4). Additionally, phosphorylated 4E-BP1

can exert a positive modulation loop by binding to rapamycin TOR kinase

associated protein (raptor) and further activating mTOR (119).

Phosphorylation of both p70S6K and 4E-BP1 are common readouts of

anabolic processes (112). Interestingly, there is evidence of a molecular

‘bridge’ (Ying-Yang 1, YY1) between mTOR and PGC-1 (120) (Figure 4).

The existence of a target such as YY1 suggests that therapeutic interventions

designed to induce muscle growth could also influence fibre type composition

and metabolism, or vice versa. However, not much attention has been paid to

investigating the potential additive effects of combining interventions to

increase muscle mass and improve metabolism.

Akt has been the focus of intense effort in developing an intervention to

enhance muscle function. For example, chemical activation of Akt protects

against sepsis in mice (121). Also, exogenous delivery of recombinant human

IGF-1 (an upstream activator of Akt) improves muscle function in dystrophic

rodents (122) and enhances the benefits of gene therapy (123). IGF-1

treatment seems safe (without side effects humans) (124) and has the

potential to improve body composition and metabolism in dystrophic patients

(125, 126). Unfortunately, there are reasons to believe that stimulation of Akt


39

via IGF-1 will not produce substantial functional improvements (71). However,

there is evidence to support that activation of Akt via TGF- signalling

inhibition could have better results (127).

Figure 4, as shown by Sandri. M et al. 2006 (128), shows how repeated muscle contraction drives the production of IGF-1, stimulating protein synthesis and fibre hypertrophy as well to down-regulating protein degradation via the Akt axis. In addition, repeated bouts of contraction allow positive changes in intracellular levels of calcium driving a muscle slow-fibre phenotype gene program which includes the up-regulation of PGC-1 . PGC-1 further down-regulates protein degradation by halting the activity of FoxO.


40

The TGF- signalling pathway

It has been over 20 years since muscle growth inhibiting proteins extracted

from injured muscles were baptised as TGF- (129). Since then, Dr. Lee and

colleagues found that the absence of a particular type of TGF- family ligand,

myostatin or growth differentiation factor (GDF)-8, is responsible for the

muscularity in Belgium Blue cattle (130). It has also been revealed that lack of

myostatin increases muscle mass across different species (Figure 5).

Therefore, is easy to understand why inhibiting TGF- signalling and

myostatin are attractive in the quest of targets against muscle wasting (131).

When present, myostatin participates in cellular differentiation (132, 133) and

inhibits growth during wasting conditions (134). Absence, (135), direct or

indirect inhibition of myostatin induces muscle growth and improves (in

rodents) diseases such as Duchenne Muscular Dystrophy (19, 136, 137),

Amyotrophic Lateral Sclerosis (ALS) (138) and cachexia (139, 140). It is also

believed that myostatin inhibition increases the success of myoblast

transplantation (19, 141).

Figure 5 shows the effect of TGF- inhibition by myostatin deletion in mice (142), cattle, dog and human (7 months old) (143).

Myostatin, as well as many other TGF- family ligands, signals through a

series of receptors and effector proteins to repress growth (142), possibly via


41

down-regulation of mitotic events and up-regulation of apoptosis. Activation of

this pathway relies on the availability of ligands such as myostatin, TGF- ,

bone morphogenic protein (BMP) or Activin. These proteins are secreted as a

pro-peptide that is later cleaved to yield the active protein (144, 145). TGF-

family ligands bind to type II and type I trans-membrane serine/threonine

kinase receptors in the membrane of the muscle cell (146). Myostatin was

originally identified to bind both the Activin type II receptor B, which can be

inhibited by Fst. Once the heteromeric ligand-receptor complex forms, the

signal propagates towards the nucleus via phosphorylation of down-stream

effector protein small mother of decapentaplegic (Smad) protein (147).

Activated Smad proteins (1-3,5, & 8) complex with co-operative Smad isoform

4 (Smad4) to regulate hundreds of gene targets, many of which are involved

in muscle growth (148) (Figure 6).

Figure 6 shows ligand binding to TGF receptors I and II to induce their activation assembling of a receptor complex that phosphorylates and activate SMAD. Phosphorylated SMAD is then joined by collaborative Smads (co-Smads) to synergistically bind to DNA and modulate transcriptional responses in the presence of cofactors, co-activators and co-repressors (149).


42

There are many ways to manipulate the TGF- pathway. For example ligand

activity/availability can be modulated via blocking the ligand with pro-peptides

(150) or with the antagonists such as growth factor-associated serum protein-

1 (Gasp-1) (151). Introduction of dominant negative receptor competitors

(149) and degradation of Smad proteins by the E3 ubiquitin ligases Smurf 1

and- 2 (152) can also deter the activity of TGF- . Inhibition of Smad2 and 3

causes myotube hypertrophy via down-regulation of protein degradation and

through mTOR dependent increments in protein synthesis (153). Increasing

the levels of the TGF- antagonist Smad7 also promotes skeletal muscle

differentiation and removes the myostatin repression over muscle growth in

vitro (154).

Therapeutic efforts to improve muscle function via inhibition of TGF- have

shown promising results. Delivery of a compound acting as an angiotensin II

receptor antagonist commonly used to treat high blood pressure, Losartan,

has shown to inhibit myostatin rendering improvements in muscle remodelling

and protection against disuse atrophy in sarcopenic mice (155). Delivery of

antibodies against myostatin showed amelioration of the pathophysiology of

dystrophies (156, 157). A similar approach is currently being tested in cancer

patients in a Phase II trial by Eli Lilly (cinicaltrials.gov identifier

NCT01505530). Delivery of pro-peptides that can bind-to and block Myostatin

in solution (150, 158) or by plasmid (159) have also shown to elicit muscle

growth and improve muscle function. Interestingly, it has been found that

inhibition of myostatin via pro-peptides may be better at improving muscle

function than anti-Myostatin antibodies (136). Encouraging results have also

been produced from delivering short interfering hairpin against myostatin. This


43

method has shown to increase muscle mass in rodents (160). Other

alternatives to manipulate this pathway are the inhibition of Activin receptor

type II B (ActRIIB) via rAAV delivery of a soluble receptor inhibitor (161), a

dominant negative receptor (141) or antibody-targeted receptor inhibitors

(162, 163). They all have shown encouraging results as a therapeutic

candidate against muscle wasting. In addition, Acceleron Pharma/Shire

developed ACE-031, a ActRIIB receptor chimera with strong capacity to

decrease body fat, increase muscle mass and improve insulin sensitivity (138,

164, 165). However, ACE-031 has been discontinued given that a phase II

clinical trial raised safety concerns (clinicaltrials.gov identifier NCT01099761).

While the safety concerns about ACE-031 have not been reported, it is

possible that this molecule had off-target effects given its lack of substrate

specificity. As shown below (Figure 7), immunoprecipitation of the ActRIIB

binds many TGF- ligands.

Figure 7 (Souza T A et al. 2008) shows specific and direct binding between ActRII- receptors and the ligands myostatin, Activin A, B, and AB, and BMP-9, -10, and -11 (166).


44

In addition to the aforementioned strategies, it is also possible to suppress

TGF- signalling via the overexpression of inhibitory binding proteins (167),

such as BMP Activin membrane-bound inhibitor (Bambi) (168, 169) and

Follistatin (170). For the future TGF- -based therapeutics, it is important to

take into account that it is important to maintain a degree of activation of this

pathway for the adequate control of cancer, reproduction and coagulation

(171-173).

Follistatin

Inhibition of TGF- ligand family members, for example by Fst, is a potential

strategy against muscle wasting. Clinical studies have shown that

endogenous Fst levels are compromised in models of muscle wasting such as

chronic heart failure (rats) (21) or chronic obstructive pulmonary Disease

(COPD, humans) (174) while the opposite effect is observed during treatment

with deacytelase inhibitors (175) – a model of muscle hypertrophy. In line,

transgenic overexpression of Fst causes pronounce muscle hypertrophy in

skeletal muscle of healthy rodents (19, 176, 177) and primates (17).

Pre-clinical studies in rodents have shown that Fst-based therapies are a

promising path against muscle atrophy. Delivery of recombinant Fst lessens

the severity of a mouse model of Spinal Muscular Atrophy (SMA) (177) and

transgenic expression of Fst (20, 178), Fst-related proteins (179) or Fst-based

myostatin inhibition (177) increased muscle mass and improved SMA and

DMD (rodents). On the down side, concerns have been raised about the

safety of Fst therapies as it may affect the reproductive system via


45

suppression of the follicle-stimulating and the hypothalamic–pituitary–gonadal

axis (180). Hence, the future of Fst relies in finding ways to deliver and

constrain its effects within muscle.

First was first isolated as an inhibitor of follicle-stimulating hormone (181).

There are two alternative splices of Fst, a long-circulating version (Fst315)

and a shorter non-circulating version (Fst288) (176)- ; the later having a

higher inhibitory capacity than its longer version (170). Structural studies have

revealed that Fst binds to TGF- family ligands (182) and facilitates their

cellular uptake and degradation (183). In skeletal muscle, endogenous

expression of complete or partial forms of Fst blocks myostatin (179) (Figure

8) and Activin (184). As mentioned earlier, this removes the inhibitory growth

effect of the TGF- signalling pathway. In turn, this seems to promote growth

through an increase in protein synthesis (185) independent of satellite cell

activation (78).


46

Figure 8 adapted from Aversa, Z. et al 2011 shows the activation of TFG- by Myostatin and the antagonists Follistatin and Follistatin Related Gene (FLRG) (186).

Potentially, Fst drives an increase in protein synthesis after activating the non-

canonical Akt/mTOR pathway (127) and its downstream target P70S6K (185).

There is evidence to support a protagonist role for the Akt pathway in Fst-

induced growth mice (127, 187). However, it is so far unknown whether

manipulation of this pathway with Fst treatment can protect against muscle

mass loss as a result of denervation or tenotomy.

It is also possible that Fst increases muscle mass via inhibition of protein

degradation. It is known that the TGF- signalling pathway control the

expression of “atrogenes” (a term referring to genes involved in atrophy,

reviewed in later section about the Ubiquitin proteasome pathway) via cross


47

talk with the Akt pathway (188). However, no studies have looked at the effect

of Fst on protein breakdown. Interestingly, there is growing evidence

suggesting that regulation of atrogenes is also under the control of PGC-1

(189-191).

PGC-1

In humans, endurance exercise progressively improves performance in long

duration sporting events by producing positive changes within skeletal

muscles (97) as well as physiological adaptations in the cardio-pulmonary,

endocrine and neuromuscular systems (192). Endurance training regulates

the expression of slow-twitch contractile fibres, mitochondria and fatty acid

usage (193-196) and also improves body composition and ameliorates the

severity of metabolic disorders (197). Endurance exercise activates a number

of transcriptional regulators as well as protein kinases that contribute to

metabolic remodelling (89). One of these key protein is PGC-1 (23, 198-200)

(Figure 9).


48

Figure 9 (left) shows a PGC-1 as the mediator between extracellular stimuli and gene regulation. On the right, wild type (WT) and transgenic (TG) mice overexpressing PGC-1 have darker red muscle colour suggesting a shift towards an oxidative phenotype (199).

PGC-1 is a transcriptional co-activator with tissue-specific activity (201) that

was first discovered by Spiegelman and co-workers (22). PGC-1 is

expressed predominantly in metabolically active tissues such as brown fat

(22, 23), liver (76, 202, 203), cardiac muscle (204-206) [reviewed in (207)],

nervous system (208, 209), cancers (210, 211) and endocrine organs (212)

but also in many other not so metabolically active (213-216).

In muscle, PGC-1 has been described as a master ‘switch’ in the control of

mitochondrial biogenesis (201, 217-219), adult skeletal muscle phenotype

(63, 96, 201, 220-223), glucose transport (224), lipid utilization (225), the

muscle’s circadian clock (226) and skeletal muscle protein turnover (189, 227-

230) [reviewed in (201, 206, 217, 231, 232)]. Increased levels of PGC-1 are

found following increased motor nerve activity, increased energy demand

(220) and cross-innervation (with slow-twitch nerves) (199). Increased levels

of PGC-1 have been associated to the beneficial effects of low (198, 233)


49

and high intensity (234) exercise and electric stimulation (235). There is

evidence to support a role for increased PGC-1 levels during muscle

hypertrophy, such is the case after treatment with beta adrenergic synergists

(236) and high protein diets (237). However, an increase PGC-1 has also

been measured during detrimental situations such as energy deprivation

(238), cachectic cytokine exposure (239), glucocorticoids and microtubule

inhibitors (230) and protein synthesis inhibitor treatments (230).

On the other hand, decreased PGC-1 levels are often found as a

consequence or in the aetiology of myopathies (99, 240, 241), high fat diet

(242), sarcopenia and age related metabolic disease (243). Down regulation

of PGC-1 is also a reflection of detrimental health conditions such as

diabetes type II [reviewed in (232)], insulin resistance (244), disuse and

denervation-induced muscle wasting (99), obesity (225), myopathies (245),

chronic obstructive pulmonary disease (COPD) (240), amyotrophic lateral

sclerosis (ALS) (246) and Duchene Muscular Dystrophy (229) suggesting that

its adequate regulation may play a key role at the onset or during the

progression of conditions accelerating muscle breakdown.

PGC-1 protein binds directly to transcription factors through lysine rich

protein interactions involving the LXXLL motif. This couples PGC-1 with most

nuclear receptors through its N-terminal (247), this relies on its nuclear over

cytosolic localization (248). In skeletal muscle, PGC-1 interacts with

estrogen related receptor alpha (ERR ) (249), peroxisome proliferator

activated receptor (PPAR) gamma (250), PPAR delta (251), Glucocorticoid

Receptor (GR) (230), Nuclear respiratory factor 1 (NRF1) (252) and Ying

Yang 1 (YY1) (120), however in other tissues PGC-1 has shown the ability to


50

co-activate other targets such as retinoid x receptor alpha (RXR ) (253)

(HeLa cancer cells) or hepatocyte nuclear factor four alpha (HNF4 ) (203) (rat

hepatocytes and mouse liver). From the aforementioned targets, it is worth

mentioning that endogenous up-regulation of PPAR delta mediates/mimics

many of the beneficial effects of PGC-1 in skeletal muscle. In particular,

PPAR delta agonists such as the ligand L165041 (254), GW0742 (255) or its

transgenic overexpression (256) have shown to increase resistance to fatigue.

Interestingly, PPAR delta has also shown to negatively regulate Myostatin

(254).

The activity of PGC-1 depends on its transcriptional regulation and post-

translational modifications. Transcriptional regulation involves the activation of

Myocyte Enhancer Factor-2 (MEF2) a target of calcineurin (257), Akt

signalling (258) and FKHR or FoxO3a (236) and cAMP-sensitive elements

(259). Post-translational modification of PGC-1 in vitro (human and C2C12

myotubes) (239, 260, 261) and in vivo (human and mice) (260-262) include

phosphorylation by the stress-activated cytokine kinase p38 (p38MAPK)

(239), by adenosine mono-phosphate kinase (AMPK) (263) and Akt (263).

The ability of PGC-1 to link skeletal muscle growth and cellular respiration is

via its interaction with YY1 (120). YY1 is a novel transcription factor common

to mTOR and PGC-1 as seen in mouse muscle and C2C12 myotubes (120).

As a consequence of exercise, ERR is a key partner of PGC-1 in the

biogenic program involving the induction of several proteins involved in

oxidative phosphorylation (249). In addition to driving an oxidative phenotype,

PGC-1 has shown a protective role against atrophy.


51

In 2006, Sandri et al. found that transgenic overexpression of PGC-1 in mice

had a protective role against denervation and starvation induced atrophy

(189). A year later, Hanai et al. showed that PGC-1 overexpression in zebra

fish and C2C12 inhibited Atrogin-1 and prevented atrophy when exposed to

statins (lipid lowering drugs known to cause skeletal fibre break down) (228).

Then, Handschin et al. (2007) reported that transgenic overexpression of

PGC-1 improves the phenotype of 5 week-old DMD mice (mdx) (229). This

was followed by Wenz et al. (2009) in a study using muscle-specific

overexpression of PGC-1 to protect mice against sarcopenia and metabolic

disease during aging (243). It is also know that myostatin-induced atrophy

produces an increase in PGC-1 levels after 7 but not 14 days, suggesting a

natural up-regulation of PGC-1 in response to atrophy (264). Furthermore,

Fuster et al. (2007) showed that cachectic mice have increased levels of

PGC-1 and a shift to an oxidative skeletal muscle phenotype (265). Also,

deletion of PGC-1 has been linked to decreased muscle fibre integrity and

recovery from exercise (219). Lastly, overexpression of PGC-1 and its

closely related protein, PGC-1 , have shown to attenuate protein degradation

(266). All together, these data reinforces the idea of PGC-1 acting in an

internal protective mechanism against atrophy.

There is also evidence suggesting that PGC-1 is not as important as many

have believed. For example, it has been found that endurance training can

lead to similar adaptation in wild type and traditional PGC-1 knockout mice

(267, 268). Similarly, deletion of PGC-1 in skeletal muscle has no effect on

exercise capacity and or mitochondrial biogenesis following endurance


52

training (269). In fact, excessive levels of PGC-1 could have detrimental

health effects such as atrophy, as shown in 2006 by Miura et al. (227).

Elucidating the potential of PGC-1 in adult muscle has proven elusive (270).

There is only indirect (pharmacological) evidence of the effects of up-

regulation of PGC-1 . The anti-diabetic compounds 5-Aminoimidazole-4-

carboxamide ribotide (AICAR) (271) and the PPAR agonist Rosiglitazone

(272) have shown to activate PGC-1 to improve energy metabolism.

However, AICAR did not prevent muscle wasting (266). Given these

evidence, it is clear that the field is in need of new models that allow testing

the effects of PGC-1 within the muscle, such as rAAVs.

Regulation of protein degradation in skeletal muscle

Muscle loss occurs when the equilibrium between protein synthesis and

degradation is broken or when the protein synthesis machinery fails to

replenish degraded proteins. Examples of situations tilting this balance are

genetic neuromuscular disorders (236), sepsis (273), fasting (274), disuse

(275-278), paralysis (275, 279) and ageing (280). Excessive break down of

proteins may also occur as an unwanted consequence of therapeutic

treatments such as corticosteroids (281-283) and lipid-lowering drugs (284).

The general consent is that degradation of muscular one or few of the

proteolytic pathways: the ubiquitin proteasome pathway (UPP) (285), the

lysosomal pathway (273), calpains (286) and the caspase (or apoptotic)

protease pathway [reviewed in (287)].


53

The ubiquitin proteasome pathway

The UPP, sometimes considered as the primary pathway for protein

degradation in skeletal muscle (114), plays a key role in the turnover of

skeletal muscle in health and disease. The UPP consists of the Ub-activating

enzyme (E1), Ub-conjugating or carrier proteins (E2s) and the Ub-protein

ligase (E3) (288). Targeting of proteins by the UPP begins with the activation

of Ub at its C-terminus by E1 (289), then Ub is transferred by one of the E2s

towards the protein of target for degradation. The transference of the Ub is

catalysed by E3 (a key enzyme conferring specificity and serving as limiting

step for ubiquination) (289). The Ub tagged protein (ubiquinated) is

recognised by a multi-catalytic complex (26S proteasome), rendering its

degradation in the proteasome (290, 291). Once a protein has been

degraded, its constituents can serve as a substrate for production of energy or

synthesis of other proteins (Figure 10 right).

Two muscle specific E3-ligases, muscle specific F-box protein

(MAFBx/Atrogin-1) and muscle RING-finger protein-1 (MuRF1), have been

identified as key players in the control of muscle atrophy [reviewed in (292)].

These proteins may also play a role in the control of carbohydrate metabolism

(293-295), suggesting they have multiple roles for the maintenance of muscle

function. There is still a lot learn from the regulation of Atrogenes. However, it

is known that nuclear factor kappa beta (NF- ) (87, 296, 297) and FoxO (87)

are two of the main signalling cascades involved in protein degradation.

NF- is a transcription factor involved in immune, inflammatory and cell

survival responses (298). Deletion of NF- has shown to increase muscle

mass, force (in fast oxidative muscle fibres), protect against atrophy and


54

enhance muscle regeneration (299). FoxO (87) is a protein controlling protein

turnover (113-115). FoxO’s activity has been found under the control of

calcineurin (300), Creb Binding protein (CBP) family p300 transcription

complex (301) and Akt (111); most probably via a negative feedback loop

(302) in models such as muscle repair (303). There is no doubt that future

therapies against muscle wasting will, in one way or another, target these

pathways and/or the UPP.

Degradation of proteins by the lysosome

The lysosome is an organelle with peptidase activity (304) of vital importance

for skeletal muscle in vivo and in vitro (114, 174, 305). The lysosome works in

three ways. Firstly, vesicles can sequester and engulf organelles or proteins

for their hydrolation in the sarcoplasm. Secondly, the lysosome can directly

consume its targets. Lastly, similarly to the UPP, a consensus peptide

sequence binds to a chaperone/co-chaperone complex that allows its

recognition for lysosomal degradation [reviewed in (306, 307)].

A key mammalian family of enzyme in the lysosome are the proteases

cathepsins. Up-regulation of these enzymes are used as markers of protein

loss (273). Although there is little evidence, it would be interesting to see what

phenotype results from interventions aimed at inhibiting parts of this pathway

during muscle atrophy. The following sections offer a summary of each of

these catabolic pathways.

The calpain-mediated protein breakdown

Calcium dependent proteinases, or calpains, are a family of cellular proteases

that are activated by increases in cytosolic levels of free calcium (308).


55

Calpains play a key role during myogenesis by intervening in cell adhesion,

migration and fusion (309). Calpains have also shown to mediate atrophy in

vitro (310) and participate transcriptional regulation in tubular cells (311).

There are at least 14 calpains. The most important are -calpain, –calpain,

calpastatin (inhibitor of the first two) and two muscle specific calpains.

Calpains ( - and -) are heterodimers containing a larger catalytic domain

and a smaller regulatory fraction (308). The main difference between -

calpain and -calpain is that the first requires millimolar and the second

micromolar changes in calcium concentration to modulate their activity (308).

In general, up-regulation of calpain activity is indicative of accelerated muscle

wasting (312-314). An exception to the rule is calpain 3a (p94). Down-

regulation of calpain 3a is responsible for some forms of dystrophy (286), a

trend that is also observed during cancer cachexia and denervation-induced

wasting (315, 316). For these reasons, calpains are another possible

therapeutic target against muscle atrophy (Figure 10 left).

The caspase pathway

Caspases are highly selective cysteine proteases that cleave proteins at

aspartic acid residues (317, 318), a fundamental property when it comes to

breaking down the actomyosin complex (319) (Figure 10 centre). The

caspase pathway is vital during pre-natal development and also plays a key

role during diseases leading to atrophy (287). An important caspase,

caspase-3, is up up-regulated in parallel to the UPP. While the UPP responds

to transcriptional regulation of atrogenes, caspase activity is increased by

apoptotic factors released from the mitochondria (320). It is believed that

activation of caspases occurs via mitochondrial and endoplasmic reticulum


56

signalling pathways (321). Disruption of this pathway has shown to protect

against atrophy and present another possibility to prevent muscle wasting

(322, 323).

Figure 10 was adapted from Powers S. et al. 2010 (324) and it shows the three main paths for protein degradation. On the left is the Calpain pathway, which is sensitive to increased cytosolic levels of calcium. The second (centre) is the Caspase mediated breakage of muscle proteins. On the right is the Ubiquitin-proteasome system for protein degradation.

It is currently unclear whether every proteolytic pathway is involved during the

breakdown of muscular proteins in different wasting conditions. Thus, it is

possible that many interventions against muscle wasting will be disease

specific. For these reasons, muscle wasting conditions have been classified in

groups and categories according to similarities.


57

Skeletal muscle atrophy, different diseases with a common outcome

Loss of myotube integrity due to protein defects often results in wasting.

Often catastrophic (325), defects in proteins such as those in contractile

apparatus or its interface with the membrane [reviewed in (326)] can be lethal.

The medical severity of these conditions will often depend on health status,

gender (327) and the type of muscle affected (189). Although most forms of

muscle wasting share similar molecular events (328), there are model-specific

differences that are important to consider when designing new therapeutics

interventions against muscle wasting (329).

The Muscular Dystrophy Association (MDA), a leading non for profit health

agency supporting neuromuscular diseases, has developed a classification for

muscle-affecting conditions (myopathies). According to the MDA, myopathies

are classified as dystrophies or disease-states (refer to www.mda.org).

Muscular dystrophies are mostly considered as inherited wasting conditions

arising from a defective protein. Below is a list containing the best known

inherited myopathies. Duchenne Muscular Dystrophy (DMD) is covered in a

subsequent section.

Motor neuron diseases are a group of inherited neurological conditions,

generally progressive in nature, that affect the nerve cells (330). ALS

and SMA are the most common forms.

Inflammatory myopathies are a group of muscle diseases that involve

inflammation of the muscles or associated tissues. These diseases

respond to a genetic predisposition or they can be inherited (331).


58

Diseases of the neuromuscular junction, or serious weakness, are

autoimmune myopathies caused by interference of the acetylcholine

receptors (332).

Myopathies due to endocrine abnormalities are normally curable and

occur in response to changes in hormones such as thyroxine (333).

Diseases of peripheral nerve are forms of inherited myopathies that

occur due to the destruction of nerves or axons that stimulate

movement (334).

Metabolic diseases of muscle are mostly inherited myopathies that

arise from the lack or malfunction metabolic enzymes or mitochondrial

genes (335).

Other non-related conditions affecting muscle tone.

Diseases-states leading to muscle wasting are conditions such as

Cancer induced atrophy (cachexia)

Any debilitating condition leading to physical inactivity including surgery

and acute illness

mechanical/medical injury to nerves or tendons (accidents, surgery and

post-synaptic block in intensive care unit)

toxins (snake bite)

malnutrition or starvation

therapeutic treatments which may have muscle wasting as a secondary

effect (dexamethasone or statins)

natural decline of muscle mass with age


59

The following section aims to show the particularities of some of the most

studied forms of muscle wasting: genetic disorders, cachexia, sarcopenia and

inactivity.

Genetic disorders

Inherited myopathies are a large group of deleterious leading to progressive

muscle loss, weakness and shorter life-span (336). The most common

myopathy, DMD (337), occurs as a result of aberrant expression (or lack) of

dystrophin. This protein anchors the sarcomere to the cell membrane (338).

DMD, first described in the late 19th Century by French neurologist Guillaume

Duchenne, has an incidence of 1 out of 3,500 live male births and a

prevalecense of 1 in 20,600 in the western world. DMD patients are born

normal, but they start experiencing muscle mass loss during their infancy.

DMD progression involves general muscle wasting, lack of postural control

and need of supportive equipment or a wheelchair during childhood. Then,

DMD patients experience cardiac, respiratory and digestive problems, which

will cause death by 20 years of age without appropriate care. As long as there

is no cure against DMD, patients will have to endure the progression of their

disease only with nutritional interventions, corticoids and palliative care.

Potential therapeutic developments against DMD have focused on rectifying

the gene mutation as well as enhancing muscle function. Promising cures are

the restoration of dystrophin via exogenous expression of the protein or parts

of it (15, 123, 339), delivery of genetic ‘fixes’ to produce a normal endogenous

gene (exon skipping) (340) or delivery of utrophin, an alternative gene that

shares functions with dystrophin (16). Of interest is the evidence supporting

the treatment of DMD patients via Fst-based therapies (19, 341, 342) or


60

overexpression of PGC-1 (229). This highlights the therapeutic potential of

these interventions.

Cachexia

Cachexia is the loss of muscle mass occurring from increased circulation of

muscle-wasting inducing factors. This is occurs in some types of cancer

(Figure 11), uraemia, and stroptozotocin-induced diabetes (328). Cachexia is

also results from chronic heart failure (343, 344), COPD (174, 345) or after

the administration of corticosteroids or blood pressure lowering medications

(284, 346). One important circulating protein involved during cancer cachexia

is myostatin (134). Myostatin can exacerbate the UPP and lysosomal

pathways while also reducing the levels of circulating anabolic hormones

(343). Together this leads to a rapid decline muscle mass and body weight.

Figure 11 from Aversa, Z. et al 2011 shows pronounced muscle wasting in a 59 year-old man with metastasizing gastric cancer. The patient had lost approximately 35% of his normal body weight during his illness (186).


61

Cancer is also accompanied by a drop in Akt phosphorylation and PGC-1

levels in skeletal muscle (347). There is a body of evidence suggesting that

treating muscle wasting is beneficial against cancer (95). A hallmark of

cachexia is that the loss of lean body mass cannot be prevented or reversed

simply by increasing nutritional intake (95). Interestingly, myostatin inhibition

has shown to protect against cachexia and increased survival (140, 348, 349).

There is also evidence suggesting that exercise mediated up-regulation of

PGC-1 can ameliorate cachexia (350).

Sarcopenia

The decline in muscle mass and decrease in muscle function as a

consequence of advanced age is known as sarcopenia (351-353). It is

controversial whether sarcopenia is a consequence or a disease. Sarcopenia

is characterised by decreased myogenic (anabolic) markers at rest and upon

load (354), increased production of oxidative species, decreased antioxidant

levels (355) and reduced capacity to activate satellite cells (353). This loss in

skeletal muscle plasticity (356) comes in hand with decreased ability to

respond to exercise (357). Myostatin and decreased Akt phosphorylation are

also present during sarcopenia (358). Interestingly, anti-myostatin

interventions have shown to protect against sarcopenia in mice (105, 359).

Also, transgenic overexpression of PGC-1 (243) or its up regulation after

treatment with Losartan (155) have shown to protect against sarcopenia.

Non-degenerative models of muscle wasting

There are several non-degenerative models of muscle wasting. These

include, for example, injury or trauma to nerves (denervation) (360) and/or

tendons. Loss of innervation leading to paralysis may occur as a result of


62

damage or death of the motor unit, sectioning of the nerve (277) or disruption

between acetylcholine and its receptor [such as a result of post-synaptic block

or neuromuscular blockade while in intensive care unit (361)]. Loss of

innervation is often used as a model to resemble the progression of

neuromuscular diseases such as ALS (362-364). In contrast to denervation,

tendon trauma leads to the mechanical unloading of a muscle while still

retaining the intact nerve that supplies with both electrical activity and trophic

factors (360).

Denervation of skeletal muscle cells is characterized by atrophy (362, 364)

and changes in morphology (365, 366). During denervation there is

accelerated protein breakdown and rapid loss in mass, probably as a

consequence of increased protein degradation (367) apoptosis (368) and

autophagy (369). Early in denervation there is nuclear translocation of FoxO3

and increased transcription of atrogenes. Later, FoxO3 is acetylated and

inhibited (370). This needs to be taken into account when designing

interventions against denervation.

Denervation is also associated with apoptosis of myofibrillar proteins (275,

371, 372) in the subsarcolemmal areas around myonuclei (373). Denervation

is followed by an increase in acetylcholine receptor density (374, 375).

decrease mitochondrial content (376), increase subsarcolemmal and inter

myobrillar concentration of oxidative oxygen species (e.g. nitric

oxide/peroxynitrite hydrogen peroxide) (368), an increase in preteoglycan

expression (377) and reduction of nitric oxide synthase (NOS) (378). NOS has

been implicated in the control of intracellular levels of calcium, myonuclei

number, satellite cell incorporation (379) and susceptibility to apoptosis (380).


63

Denervation is often accompanied by DNA fragmentation (368) and occurs in

parallel to deleterious changes in transcription factors. These includes the fall

in both Transcription factor A (Tfam) and PGC-1 , as well as an increase in

the ratio of between the anti-apoptotic B cell lymphoma 2 associated x protein

(Bax) and the pro-apoptotic B cell lymphoma 2 protein (Bcl-2). In addition,

denervation has been shown to put a break in the cell cycle (in laryngeal

muscle of rats) (381) and increase the levels of Myogenic regulator factor D

(MyoD), cyclin-dependent kinase inhibitor p21 and retinoblastoma protein. Not

all of these changes are associated with muscle atrophy. It is possible that

some responses may correspond to a natural protective response against

denervation (382) [such as flavoprotein apoptosis-inducing factor (AIF), a key

anti-apoptotic molecule (383)]. During denervation-atrophy there is also a fall

in phosphorylation of tumour protein 53 (p53) (384). Interestingly,

overexpression of p53 improves aerobic exercise capacity and augments

skeletal muscle mitochondrial DNA content by interacting with Tfam (385).

Despite the vast understanding about denervation-atrophy, there is no

therapeutic intervention against this form of muscle wasting. Denervation

could act as a surrogate model to understand the effects of immobility, neuro-

degeneration and accidental nerve/tendon damage. And testing therapeutics

interventions during denervation could be used to assess the role of nerve-

trophic factors during muscle growth.

Potential interventions against muscle wasting

As previously discussed, there is a large unmet medical need for an

intervention to maintain or re-establish skeletal muscle mass and function.

Current treatments to reduce the loss of muscle mass such as nutritional


64

supplementation (providing increased calorie intake and special amino acid

compositions) and hormonal treatment (e.g. growth hormone, testosterone,

IGF-1, and insulin) have been largely ineffective [reviewed in (386)]. It would

be cost efficient to develop a ‘one for all’ treatment than targeting each

disease independently. Denervation-induced atrophy offers a model fit for the

porpoise of testing these interventions.

Denervation-atrophy is characterized by the loss in nerve activity and

paralysis. Many interventions have shown promising results against

denervation-atrophy. Some of the most successful results have been obtained

with electrical stimulation or pharmacological/genetic interventions that

mimicking endurance or resistance training. The following section summarizes

some of these known approaches and explores the potential of novel

interventions.

Electrical stimulation

Denervation increases protein degradation but does not interfere with the

ability to synthesize proteins (365). For this reason, mechanical interventions

that classically increase protein synthesis can effectively restore muscle mass

and function in this model of atrophy. Such is the case of synergistic tenotomy

which increases muscle mass by 40% within 6 days in healthy mice (387) and

similarly (but to a lesser extent) increases mass of denervated rat muscle

(Figure 14). This showed that denervated muscle can grow even during

absence of neuronal influences (388). Stretching muscle fibres has also

shown to increase the cross sectional area of denervated soleus muscle

fibres (see Figure 4), a process involving activation of Akt signalling but not

mTOR (389). The most successful therapeutic intervention against


65

denervation has been electrical stimulation. Electrical stimulation can maintain

mass and force production of skeletal muscle after denervation in rats (390),

dogs (391) and humans (392). Perhaps the future awaits with implantable

stimulation devices to preserve muscle mass and force, a successful

approach to maintain function in the EDL of young (393) and old rats (394).

Figure 12 shows how muscle contraction protects skeletal muscle against denervation in rats after repetitive stretch in denervated soleus (A) (389) and electrically contracted denervated EDL (390) (Cd stands for contractions daily).

Synthetic compounds and transgene manipulation

Pharmacological compounds have been shown to protect against

denervation-atrophy. For example, systemic administration of -adrenergic

receptor agonist has an anabolic effect that reduces the muscle loss by 15%

in denervated rats. This protection seems to be rapamycin insensitive (395),


66

an possibly Akt independent (396). The administration of proteasome

inhibitors, which block the UPP, can also attenuate denervation atrophy by as

much as 50% in rat muscle (291, 397).

Transgenic manipulation of genes has also shown promising results against

denervation (Figure 13). A 10 % protection against atrophy in the

gastrocnemius muscle of denervated mice has been observed in mice lacking

Bax (322). Bax is an apoptotic protein that induces DNA fragmentation,

mitochondrial mediated apoptosis, activation of caspases, increases oxidative

stress and inhibits the apoptosis repressor with caspase recruitment domain

(ARC) in mice (398). Transgenic deletion of caspase-3, another example of a

pro-apoptotic target involved in muscle wasting, spares muscle wasting by

10% in denervated mice. Unfortunately, it is unlikely that sustained

suppression of caspases will become a clinical application because this could

interfere with the normal turnover of proteins required for a health (320).

Another potential approach is to block the active form of I-kappa beta (I )

kinase (Ik kB) and in turn de-activate NF- signalling and expression of E3

ubiquitin ligases (399). Although genetic and pharmacological suppression of

this pathway does not produce an inherent phenotype, inhibition of NF- in

transgenic mice reduces atrophy in the gastrocnemius by around 13% after 2

weeks of denervation as well as protecting against other models of wasting

such as cancer cachexia. Similarly, effects have been found after the

inhibition of NF- signalling components (299, 400) [for comprehensive

review of this pathway refer to Mourkioti et al. (297)] highlighting this pathway

as a potential therapeutic targe. This is further supported by the evidence

showing that Tumour Necrosis Factor (TF) like weak inducer of apoptosis


67

(TWEAK), a key cytokine related to NF- activation, plays a role in

denervation. Transgenic overexpression of TWEAK leads to a decrease in

muscle size and it exacerbates denervation-atrophy (predominantly in fast

fibres). This occurs via activation of NF- and its target MuRF1. Ablation of

TWEAK (genetic or chemical blockade) increases muscle size and rescues

the loss of mass and strength after denervation (401).

The Akt pathway is another potential target against denervation-atrophy.

Increasing IGF-1, an upstream regulator of Akt, reduces atrophy by 20% after

two weeks of denervation (111). A similar effect is obtained by increasing the

activity of Akt (104). Crossing of mice carrying active Akt and lacking Inhibitor

of NF- (IKK2) have shown further improvement in the protection against

wasting (299). However, the benefits IGF-1 treatment could decrease with

age (402).

PGC-1 overexpression can partially protected against muscle wasting (243).

PGC-1 has been shown to protect against denervation via modulation of

FoxO3, conferring up to a 30% protection in denervated muscles. Although

the exact mechanisms remain elusive, PGC-1 seems to drive a genetic

program with the capacity to confer protection against atrophy via down-

regulation of E3 ubiquitin ligases (189).


68

Figure 13. Genetic interventions that protect against denervation-atrophy. Adult overexpression of a constitutively active form of Akt (104) (A), transgenic (Tg) expression of a muscle IKK super repressor (MISR) (399) (B), transgenic deletion (KO) of caspase-3 in the gastrocnemius of mice (323) (C), transgenic deletion of the Bax gene (322) (D), transgenic muscle specific overexpression of PGC-1 (189) (E) and transgenic deletion of TWEAK protein in mice (401) (F).


69

The TGF- signalling pathway has also shown to participate in denervation

atrophy. The absence of nerve supply evokes an increase in myostatin levels

(active form) in both rats (403) and mice (404). Accumulation of myostatin in

denervated muscles is reduced with exercise (405), suggesting that myostatin

inhibition could potentially buffer the effects of denervation. Interestingly, as

myostatin levels increase during denervation so does the phosphorylation of

mTOR, suggesting that skeletal muscle has mechanism in place to defend

from atrophy (406). However, it is not known whether post-natal inhibition of

myostatin via, for example, treatment with Fst could protect against

denervation atrophy.

An innovative approach, increasing muscle mass and fatigue resistance

Regular exercise has great health benefits, including healthy aging and

improved physical performance. While reduced physical activity/exercise,

muscle disuse or disease has the opposite effect. In general, exercise is

believed to improved strength and/or endurance. Training to increase strength

involves the use of resistance to muscular contraction and increases force,

anaerobic endurance and skeletal muscle mass (388). While training to

improve endurance improve stamina, exercise efficiency and resistance to

fatigue with little or no change to muscle mass (193). Separately, endurance

and resistance training have positive health effects (407). But in combination

(concurrent training), endurance and resistance training may even better

(408). Concurrent training has been shown to improve energy metabolism

(409) [a response preserved in the elderly (410, 411)] and be best suited to

improve the health sedentary people (412) over the performance of highly

trained athletes (413, 414).


70

Mimicking the effects of concurrent training is a very palatable idea from a

therapeutic point of view (415). However, it is currently unknown whether it is

possible to make a muscle fatigue resistant and strong at the same time

(416). For this reason, it would of interest to test if post-natal manipulation of

each PGC-1 and Fst or their combination could reproduce subset

adaptations of endurance and resistant exercise.


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Skeletal muscle metabolism

Skeletal muscle makes up to half of the body weight in non-obese humans

and is fundamental in metabolic homoeostasis (417, 418). The regulation of

skeletal muscle mass and metabolism is both complex and depend one on

each other. There are two main nutrients involved in energy production,

glucose and lipids. Hence, their correct regulation it is fundamental for muscle

function.

Glucose metabolism

Glucose is one of the main energy substrates. Uptake of glucose by skeletal

muscle relies on the translocation of glucose transporters to the sarcolemma

and the t-tubules (419) [reviewed in (420)]. In muscle, glucose Transporter 1

(GluT-1) is responsible for maintenance of basal glucose uptake while

Glucose Transporter 4 (GluT-4) acts upon the increase in levels of insulin

concentration and/or muscle contraction (421, 422). Once inside the cell

glucose trapped inside the cell by its phosphorylated by hexokinase or it is

stored as glycogen. Glycogen and glucose generate energy via anaerobic and

aerobic metabolism. Anaerobic metabolism occurs at the expense of

generation of deleterious molecules such as lactate and hydrogen while

complete oxidation glucose occurs through aerobic respiration (423).

Insulin-dependent glucose uptake is a process that begins with the binding of

insulin to the insulin-receptors located in the membrane of skeletal muscle

cells. This renders a conformational change in the insulin receptor that

activates the kinase domain in its intracellular portion. The intracellular

domains initiates a phosphorylation cascades via activation of the Insulin

Receptor Substrate 1 (IRS-1) which in turns activates PI3K rendering the


72

phosphorylation of it downstream target Akt1 at Thr308 and Ser473 (424).

Activation of Akt triggers the translocation of GluT-4 (425) as well to signal

downstream through activation of mTOR/p70S6K (426). This links glucose

uptake with muscle growth (427). Akt exerts control of metabolism at multiple

levels. In healthy individuals, the Akt pathway is constantly fluctuating

between active and inactive. In contrast, insulin insensitive postpartum

women present constant activation of this signalling cascade (428). Akt also

controls FoxO, a key transcription factors involved in atrophy and in the

negative regulation of the insulin related receptor. It still remains to be fully

elucidated whether post-natal interventions that activate Akt to induce muscle

growth can also increase insulin sensitivity.

Increased glucose uptake can also occur independently of insulin via

activation of GluT-4 in response to muscle contraction. This mechanism

increases allows to compensate for increased energy demands (429). This

process involves two mechanisms [reviewed in (430)]. The first, augmenting

the intracellular concentration of calcium directly up-regulates GluT-4

translocation (431). The second, a drop in the ATP to Adenosine

monophosphate (AMP) ratios as a consequence of decreased in fuel

availability activates AMPK which in turns increases glucose uptake (432).

An increased in glucose uptake is also observed during skeletal muscle

growth. It is possible that this effect is linked to higher net calcium uptake,

more contractile units and increased energy storage capacity. And key

signalling cascade that could be behind these changes is the TGF- pathway

(6, 433, 434). There is evidence from obese and insulin-resistant human

subjects to support an increase in the activity of the TGF- pathway during


73

these conditions (435, 436). This pathway inhibits muscle growth and this

could in turn decreased glucose uptake during atrophy (245, 437). On the

other hand, inhibition of the TGF- family ligand myostatin is associated with

improved insulin sensitivity during high-fat diet induced obesity (6, 438-440).

Similarly, mice lacking Smad3 (a TGF- signalling component) are protected

against insulin resistance (441). The rationale is that TGF- pathway inhibition

increases Glut-4, Akt signalling and AMPK driven up-regulation of metabolic

regulators such as PGC-1 (442). However, it is currently unknown whether

post-natal delivery of the TGF- pathway inhibitor Fst can have a positive

influence over glucose metabolism.

Lipid metabolism

Lipids are a broad group of molecules that play key roles in cellular structure

and energy metabolism. The process of lipid metabolism includes the

breakdown of adipose and intramuscular triglyceride, the transport of free fatty

acids (FFAs) from adipose to muscle, the entry of FFAs into muscles from

blood, activation of FFAs, entry of activated FFAs into mitochondria, Beta

oxidation ( -Oxidation) of FFAs and mitochondrial of oxidation (443).

Oxidation fatty acids is the most efficient source for skeletal muscle ATP

production (444). Triglycerides are mainly used at rest and during low

intensity exercise by mitochondrial-rich muscles (443). Endurance training or

endurance like interventions, such as PGC-1 overexpression or PPAR

agonists, have the ability to improve triglyceride metabolism (195, 198, 233,

272, 440, 445, 446). Muscle atrophy is associated with the opposite effect

(379, 447, 448). Recently it has also been shown that mice over-expressing

Akt isoform 1 (Akt1), post-natal myostatin blockade and transgenic deletion


74

myostatin, all results in increased muscularity and reduced fat content when

challenged with high fat diets (440, 449). This is likely to occur in order to

improved energy metabolism to support growth as well as an increased

efficiency of the myofiber membrane to transport nutrients (450).

There is rodent evidence to support that changes in the membrane lipidome

of skeletal muscle regulates energy metabolism (451). In humans, changes in

diet (452) and exercise can regulate the lipidome (453, 454). As described by

Andersson, A et al. in 1998, low intensity exercise seem to reduce the

phospholipid proportion of palmitic acid (16:0) and linoleic acid [18:2(n-6)] and

the sum of n-6 fatty acids [18:2(n-6), 20:3(n-6), 20:4(n-6)], whereas oleic acid

[18:1(n-9)] increases by training while the fatty acid profile in skeletal muscle

triglycerides remains unchanged. However, these observations have not been

proved true. It is possible that lipidomic adaptions precede, follow or mediate

many changes in skeletal function. Perhaps muscle growth, such as it occurs

with Fst treatment, has unknown effects on the lipidome. However, this will

remain unknown until such experiment is performed. Testing potential

therapeutics interventions to improve different aspects of muscle function

could lead to better therapies. Growing pressure to come up with new

therapies, stricter regulatory legislations and scientific advances require new,

innovative, specific models for the development of therapies. Fortunately, new

gene manipulation technologies are helping in the understanding of skeletal

muscle function. A particular technology, rAAV viruses, has risen above the

rest due to the potency, tissue-specificity and lack of immunogenicity. Clearly,

these viruses hold great potential for the development of therapies in the new

millennia (15, 16).


75

Gene therapy with rAAV

Gene therapy allows the introduction of new genetic material to correct gene-

malfunctions, re-/introduce new/missing genes or depressed endogenous

transcripts. During the last 15 years, Recombinant adeno-associated viral

vectors have generated much optimism as the preferred gene therapy

platform for human use. rAAVs have been safely tested in clinical trials

involving over 300 patients (211, 212) (see table 1 for a list of trials). The

reason why rAAVs are so attractive is because they are non-replicative, non-

integrative, non-pathogenic and generally safe (455). rAAV could allow to

tissue specific delivery (456) of one or multiple genes at once. According to

the United States of America Department of Health and Human Services

Agency for Food and Drug Administration (FDA), risks associated with

delivery of rAAVs are minimum (457). While the approval of therapies based

on rAAVs by the FDA is still in progress, their uptake as gene delivery tools

for research in vivo has grown rapidly.

76

Table 1 [adapted from High and Aubourg (18)] shows a current list of registered clinical trials using

Disease Transgene Serotype

Route of administration

Clinical trial (number of patients)

1-Antitrypsin deficiency (21)

1-Antitrypsin rAAV-2, rAAV-1

Intramuscular (IM) Phase I/II

Age-related macular degeneration (recruiting)

Placental Growth Factor Fusion protein

rAAV-2 Intravitreal injection Phase I

Alzheimer’s disease (8) NGF rAAV-2 Intracranial Phase I/II

Arthritis (127) Tumour Necrosis Factor Receptor Fusion Protein

rAAV-2 Intraarticular Phase I

Batten disease (~10) Tripeptidyl-peptidase I rAAV-2 intracranial Phase I

Canavan’s disease (10) Aspartoacylase rAAV-2 Intracranial Phase I

Cystic fibrosis (>130) Cystic Fibrosis Transmembrane conductance regulator

rAAV-2

maxillary sinus, bronchoscopy to right lower lobe, aerosol to whole lung

Phase I/II

Severe heart failure (9) Sarcoplasmic reticulum Ca2+ ATPase

rAAV-1 coronary artery infusion

Phase I/II

77

Hemophilia B (8 + 8) Coagulation Factor IX rAAV-2 IM and hepatic Phase I/II

HIV vaccine HIV virus protein gag rAAV-2 IM Phase I/II

Liprotein Lipase deficiency (8)

Lipoprotein Lipase rAAV-1 IM Phase I/II

Lebel's Congenital amaurosis (blindness)(12 + 3 + 3)

Retinal Pigment epithelium-specific 65Kda protein

rAAV-2 Subretinal Phase I/II

DMD (3) Microdystrophin rAAV-1/2 hybrid

IM Phase I

Muscular dystrophy: Limb Girdle (~6)

alpha-Sarcoglycan rAAV-1 Repeated IM Phase I

Parkinson’s disease (22) AA decarboxylase, GA decarboxylase, Neutrophin

rAAV-2 Intracranial Phase I/II

78

Pompe’s disease (recruiting subjects)

Alpha-glucosidase (GAA)

rAAV-1 intradiaphragmatic Phase I/II

Becker Muscular Dystrophy and Sporadic Inclusion Body Myositis (15)

Follistatin (circulating form)

rAAV-1 IM Phase I


79

rAAVs classify as a class of Dependoviruse (family Parvoviridae, subfamily

Parvovirinae). They have a single stranded DNA viral genome of about 4.7kb

packaged in a 25nm protein capsid (458) (Figure 14). The rAAV genome is

composed of open-reading frames encoding replication and capsid proteins flanked

by inverted terminal repeats (ITRs) at either end of the viral genome. There are

mainly 9 rAAV serotypes, each with variable tissue tropism (459). These serotypes

share similar genome size and organization, however they differ in the amino acid

capsid (459).

Figure 14 shows (A) a viral particle, (B) the representations viral capsid and transgene and (C) the transgene constituents (promoter, transgene cDNA and polyA signal).

Tissue specific uptake of rAAVs is controlled by capsid serotype and transgene

expression by the promoter. In combination, capsid and promoter allow tissue

specific, stable delivery of genes into muscle and other tissues in rodents (460),

canines (340, 461), macaques (17, 462) and humans (463). Cellular uptake of

rAAVs requires its binding to rAAV receptors on the surface of the target cell (464).

Then, endocytosis of rAAVs into the nucleus occurs through vesicular trafficking,

endosomal escape and nuclear transport. Once in the nucleus, rAAVs particles shed

their capsid to delivery its transgene (Figure 15).


80

Serotype 6 rAAV (rAAV6) are a favourite to treat skeletal muscle. Single delivery of

rAAV6 has shown to cause body-wide transduction of the entire skeletal musculature

(465). Body-wide delivery of rAAV6 has been successful at treating mice (339) and

canines (466). Overexpression of insulin and glucokinase via delivery of rAAV6 has

shown improve glycaemia in diabetic mice (467). These characteristics position

rAAVs at the forefront of therapeutic tools when focusing on enhancing muscle

function.

Figure 15 (Ding et al. 2005) represents the steps of rAAV transduction including: (1) viral binding to a receptor/coreceptor, (2) endocytosis of the virus, (3) intracellular trafficking of the virus through the endosomal compartment, (4) endosomal escape of the virus, (5) intracellular trafficking of the virus to the nucleus and nuclear import, (6) virion uncoating, and (7) viral genome conversion from a single-stranded to a double-stranded genome capable of expressing an encoded gene (464).


81

Aims and hypothesis

The outstanding ability of skeletal muscle to respond to environmental stress (468)

plays both in favour and against its function. There is hope that manipulating

signalling cascades to resemble exercise may outweigh disease. Two possible

interventions to do this and improve skeletal muscle function are down-regulation of

TGF- signalling by increasing Follistatin levels and up-regulation of PGC-1 . Thus,

the general aims and hypothesis of this research will be as follow:

Aims

1. To test the hypothesis that Follistatin overexpression can prevent (or treat) non-

degenerative muscle wasting (nerve and/or tendon injury) by increasing muscle

growth.

2. To test the hypothesis that Follistatin-induced muscle growth will increase the

demands and utilization of glucose and lipids.

3. To test the hypothesis that muscle growth and fatigue resistance driven by

overexpression of Follistatin and PGC-1 , are not mutually exclusive in the control of

muscle mass and function.

Hypothesis

1. It is hypothesised that Follistatin will affect signalling pathways that increase

protein synthesis and decrease protein degradation. In turn, this will have a

protective effect against models of wasting such as denervation. In addition, it is

expected that denervation will exacerbate this pathways and that Fst will buffer this.

2. It is hypothesised that Follistatin overexpression will produce muscle growth. This

will in turn increment the demands for glucose and fatty acids.


82

3. It is hypothesised that co-expression of PGC-1 and Follistatin will have additive

effects in increasing muscle mass and enhancing force production capacity.

Implications of this work

This work will show whether Follistatin can be used to treat non non-degenerative

muscle wasting. Also this thesis will demonstrate the effects of Fst-induced growth

on glucose and lipid metabolism. Finally, this research will be the first to show the

effect of post-natal PGC-1 delivery and the effect of its combined delivery with Fst

on muscle mass and function. This work will create a novel body of knowledge.


83

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