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General Introduction

1.1 General introduction

Skeletal muscle comprises a complex matrix of individual units of force

generating protein, singularly ensheathed within tubular lipid membranes, providing

tensile strength of protein, complimented by passive elasticity of lipid in a synergy of

fast excitatory stimulation and dynamic adaptive plasticity. The muscle fibre‟s

compressive force generation potential is however not a reflection of toughness or

resilience of tissue and, when separated from one another, muscle fibres are indeed

brittle and easily damaged by bending and compression forces. Moreover, significant

damage to the sarcolemmal membrane surrounding individual fibres is sufficient to

cause necrotic death of the fibre. Hence, the majority of research in the field of

muscle diseases focuses on the proteins located at the interface of membrane and

force generating protein. The mechanisms underlying the dynamic adaptability of

fully differentiated adult skeletal muscle remain largely unknown, providing an

attractive arena for the investigator. The discoveries that have been made largely

stem from the fundamental theory that differentiated muscle arises from the fusion of

mononucleate cells during development, giving rise to the classical multinucleate

fibre structures, commonly associated with muscle. Indeed, it was the discovery of

the satellite cell (Mauro, 1961) that underpinned the portrayal of differentiated muscle

as maintained and plasticised through the dedicated currency and conveyance services

of a resident population of mononuclear myogenic precursor cells located beneath the

basal lamina of mature muscle fibres, at the interface of membrane and functional

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protein. The complexity and diversity associated with this anatomically distinct and

intricately dynamic population of cells has belayed the occupations of the skeletal

muscle biology field for nearly 50 years, and continues to feature heavily at the

forefront of research into skeletal muscle disorders.

1.2 Historical perspective

„Muscular dystrophy‟, from the Latin, „dys‟, meaning difficult and the Greek

„trophe‟, relating to nourishment, denotes a heterogeneous group of neuro-muscular

diseases, the most common of which is Duchenne muscular dystrophy (DMD). Dr.

Guillaume-Benjamin-Amand Duchenne, who‟s name is born by more than 10 of the

now recognised list of over 40 such conditions, was born Sept.17, 1806, in Boulonge-

sur-Mer (Rudolf Kleinert). As well as a medical practitioner, his hobby was studying

the emerging sciences of neurology and electrical stimulation, for which he famously

constructed an apparatus allowing the surface-electrode stimulation of bodily tissues

from living subjects. His scientific persuasions were complemented by an interest in

photography, and he was one of the first people to use photographs instead of

drawings to document pathological conditions for medical reference. In a series of

works published between 1861-1872 (Paraplégie Hypertrophique de l‟enfance de

cause cérébrale, De l‟Electrisation localisée et de son application à la pathologie et à

la thérapeutique and L'Electrisation Localisée; http://www.whonamedit.com),

Duchenne described a collection of juvenile patients with conditions that he

considered to represent a form of juvenile spinal muscular atrophy, characterised by

increases in the fatty interstitial tissue surrounding the musculature. He is also

thought to be responsible for devising the first implement and procedure for

conducting muscle-derived biopsy from living patients without anaesthesia (Charriere

and Duchenne, 1865), there by enabling him to demonstrate the progressive nature of

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the disease. Although now widely regarded as the first person to document the

conditions which now bears his name, Duchenne may actually have been studying a

condition already described in detail by an English physician by the name of Edward

Meryon (Meryon, 1852, reviewed by (Emery and Emery, 1993). Meryon described a

condition which particularly impressed him due to its “predilection for males and its

familial nature”, which presented the now familiar symptoms of difficulty in walking

from an early age and later climbing stairs, with eventual loss of ambulation and death

in the teens. Following necropsy of an affected 16 year old boy, he commented “The

chief structural change existed in the system of the voluntary muscles, which was

throughout the entire body atrophied, soft, and almost bloodless… the striped

elementary primitive fibres were found to be completely destroyed, the sarcous

element being diffused, and in many places converted into oil globules and granular

matter, whilst the sarcolemma or tunic of the elementary fibre was broken down and

destroyed" (Emery and Emery., 1993). The parallels with modern-day descriptions of

dystrophic tissue are obvious and Meryon‟s work was actually discussed academically

by Duchenne, who failed/refused to recognise the similarities in his own work, instead

choosing to consider Meryon‟s studies to describe a more specific form of muscular

atrophy than his own findings. Indeed, many medical descriptions exist preceding

either man‟s contribution but it is Duchenne who retains much of the credit for the

early work on characterising this class of disease. These diseases are now collectively

referred to as neuro-muscular disorders, or muscular dystrophies, of which DMD is

the most common.

1.3 Clinical presentation

DMD patients typically present clinically at 4-5 years of age, with symptoms

including difficulties in standing from a seated position, climbing stairs, and keeping

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up with their peers during play. The muscle degeneration is progressive and

replacement with fibrous/fatty tissue occurs, which ultimately results in muscle

contractions and eventual death by cardiac or respiratory failure by late teens to mid

twenties (Emery and Walton, 1967). The introduction of positive-pressure ventilation

has since increased life expectancy into late-twenties and early thirties. Severe

cognitive impairments such as reduced verbal skills and delayed reading learning are

seen in approximately 30% of DMD cases (Moizard et al., 2000). Elevated levels of

creatine kinase derived from muscle, in serum and amniotic fluid (Emery, 1977)

remain the most widely used clinical markers in DMD diagnosis, combined with

mutational analysis based DNA sequencing tests for patients and their families, where

possible. DMD displays an X-linked inheritance pattern and appears in

approximately 1 in 3,500 males (Emery, 1991). Females are usually heterozygous

carriers. Their skeletal muscles are multinucleate mosaics of affected and unaffected

cells due to random X-inactivation in muscle precursor cells, where compensatory

dystrophin expression is sufficient to render a normal phenotype. However, female

carriers have been shown to develop cardiomyopathy (Hoogerwaard et al., 1999a,

Hoogerwaard et al., 1999b), which highlights a physiological difference between

skeletal and cardiac muscle, where the latter is thought to comprise around 75%

mononucleate post-mitotic cells (Olivetti et al., 1996) and the former contains a small,

yet indispensable, population of resident stem cells/satellite cells [1-6% of total

myonulcei (Allbrook, 1981)], possessing high myogenic potential (Jackson et al.,

1999). Becker‟s muscular dystrophy (BMD) also arises from mutations in the DMD

gene, although phenotypes are milder due to the in-frame nature of the mutations,

which result in differing levels of expression of partially functional, truncated

dystrophin proteins (Hoffman et al., 1987, Hoffman and Kunkel, 1989).

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1.4 Molecular principles of DMD

Genetics and molecular biology studies have facilitated the elucidation of the

DMD gene carrying the mutations responsible for the DMD phenotype (Monaco et

al., 1986) and its encoded protein product, dystrophin (Hoffman et al., 1987). Loss or

truncations of this protein are responsible for the onset of DMD, and the milder BMD,

respectively (Hoffman et al., 1987, Hoffman and Kunkel, 1989). Sequence analysis

of the dystrophin gene revealed homology to the spectrin super-family (Koenig et al.,

1988) and using the best available spectrin sequence data available at the time, that of

Drosophila melanogaster, the NH2-terminal domains of -spectrin, -actinin, and

dystrophin were found to contain striking sequence conservation, thought to reflect

conservation of structural and functional properties in these proteins (Byers et al.,

1989). The structural similarities of spectrin and dystrophin, mainly the conserved

actin binding NH2 domain, combined with the sub-membrane distribution of

dystrophin (Zubrzycka-Gaarn et al., 1988, Beam, 1988, Arahata et al., 1988), lead to

the idea that dystrophin may protect the integrity of the muscle cell sarcolemma

against muscular contraction in a similar fashion to the manner in which spectrin

allows erythrocytes to become elastic when passing through narrow capillaries (Byers

et al., 1989).

Dystrophin is the largest known gene covering 2.6Mbp within the Xp21 and

contains 79 exons - it represents some 0.1% of the entire human genome. In normal

skeletal muscle the dystrophin protein derived from full length transcription of this

gene (which results in the mature mRNA of 14 kb) has a molecular weight of 427kDa.

However, since the initial discovery, it quickly became apparent that the dystrophin

gene is expressed as several tissue specific isoforms from multiple promoters residing

within the gene. In decreasing order of mRNA length, the nine known isoforms of

dystrophin in the human genome are: Cortical or brain dystrophin; Dp427c (Nudel et

al., 1989), Purkinje dystrophin; Dp427p (Gorecki et al., 1992), muscle dystrophin;

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Dp427m (Koenig et al., 1987), lymphocyte dystrophin; Dp427l (Nishio et al., 1994),

although the significance of this promoter has since been disputed (Wheway and

Roberts, 2003), retinal dystrophin; Dp260 (D'Souza et al., 1995), CNS dystrophin;

Dp140 (Lidov et al., 1995), Schwann cell dystrophin; Dp116 (Byers et al., 1993), and

also the ubiquitously expressed apo-dystrophin isoforms - Dp71 (Bar et al., 1990) and

Dp40 (Tinsley et al., 1993) (Figure 1.4.1; A). An additional level of complexity

exists due to alternative splicing of all known dystrophin transcripts, where both the

5‟ (Surono et al., 1997) and 3‟ (Nakamori et al., 2007) regions have been shown to be

alternatively spliced in human and mouse skeletal muscle (Figure 1.4.1; B and C,

respectively).

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Figure 1.4.1 Predominant promoters and products of the DMD gene. Schematic

illustrating promoter locations (upper panel; bent arrows) and main protein products (lower

panel) of the DMD gene in humans and mice (A) with splicing products identified in the 5‟

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(B) and the 3‟ (C). Exons are numbered and in-frame variants depicted by bold lines, out of

frame by dashed lines (B and C).

Specific functions remain to be ascribed to these specific protein products

derived from the dystrophin gene. Sequence-based structural predictions led to the

now widely accepted proposal that Dp427m is a rod-shaped protein consisting of four

domains: an N-terminal actin-binding domain, twenty four triple helix spectrin-like

repeats separated by four hinge regions, a cysteine-rich domain containing two

predicted calcium binding motifs, and a unique C-terminal domain (Koenig et al.,

1988). It is perhaps more suitable to describe dystrophin in terms of its location

within normal muscle, rather than to attempt to assign any defined function, as

consensus opinion views dystrophin as a cytoskeletal linker molecule, located in

distinct sub-sarcolemmal bands distributed evenly along differentiated muscle fibres,

where it shares links with F-actin at its N-terminus, and proteins such as nitric oxide

synthase (NOS) via the syntrophins, the dystrobrevins, and syncoilin at its C-terminus

(Suzuki et al., 1994). However, perhaps the most well known link regarding DMD is

that made between the cysteine rich domain and the dystrophin glycoprotein complex

(DGC) or dystrophin associated protein complex (DAPC), where dystrophin appears

to coordinate a link from the actin cytoskeleton right through to laminin-2 of the

extracellular matrix (Ahn and Kunkel, 1993, Campbell, 1995) by way of -

dystroglycan (Jung et al., 1995), the sarcoglycans and sarcospan located in the

sarcolemma, and -dystroglycan on the external face of the membrane (Figure 1.4.2)

(Yoshida and Ozawa, 1990, Ervasti and Campbell, 1991). Mutations within

dystrophin or almost any other component of the DAPC complex have been shown to

give rise to various types of muscular dystrophies (reviewed by Khurana & Davies,

2003), yet the precise mechanisms relating to each pathology remain largely elusive,

as do the specific functions of the individual proteins involved. One such protein,

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which has recently acquired evidence of function is -sarcoglycan (adhalin), a

constituent of the four-membered sarcoglycan complex which is retained in the

DAPC through its link to -dystroglycan. Mutations of this gene in humans produce

the phenotype referred to as Limb Girdle Muscular Dystrophy type 2D (LGMD 2D),

which shares similarities with DMD. -sarcoglycan has been shown to not only bind

ATP (Betto et al., 1999), but also to degrade it, and the kinetics for this event have

been suggested to be evidence of its function as an ecto-ATPase enzyme (Sandona et

al., 2004). Considering the loss of DAPC complex in the absence of dystrophin, this

could suggest the loss of some degree of inherent ATP-hydrolysing potential from

dystrophic muscle.

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Figure 1.4.2 Structure of the Dp427 protein and its interactions with other components

of the dystrophin associated protein complex (DAPC) at the sarcolemmal membrane of

skeletal muscle. Illustration depicting the main structurally distinct domains of the full

length Dp427 protein. N-terminal actin-binding domain: Calponin homology domain 1

(CH1); cyan, CH2; green. Central rod domain: 24 coiled-coil, triple helical spectrin-like

repeats. Cysteine-rich region: WW domain (containing two highly conserved tryptophan

residues); green, Calcium binding EF hands; red, orange, cyan and purple. Carboxy-terminal

region: syntrophin-binding element; yellow, and leucine zipper domains; red (upper panel).

Dystrophin (red) facilitates anchoring of DAPC complex members via laminin in the

extracellular matrix, through the dystroglycan complex, to the intracellular contractile

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machinery. DAPC members include: - and -dystroglycan (DG, DG), sarcospan (spn),

-, -, - and -sarcoglycans (SG), syntrophin (syn), - and -dystrobrevin. Syncoilin,

dysferlin, calpain-3, caveolin-3, filamin-c, Grb-2, and nNOs all interteract with the DAPC

orchestrating downstream signalling cascades.

Sequence homology searches using cDNA libraries from humans, sea urchins

(Strongylocentrotus pupuratus), fruit flies (Drosophila melanogaster), electric rays

(Torpedo californica) and nematode worms (Caenorhabditis elegans) have identified

multiple genes showing relation to dystrophin, prompting investigations into the

evolutionary origin of the gene, which is thought to be highly conserved throughout

metazoans, implying a fundamental role in animal biology (Roberts and Bobrow,

1998). Evidence for this has been provided recently with the demonstration that the

membrane-cytoskeletal function of dystrophin extends to maintaining neural integrity

in C. elegans (Zhou and Chen, 2011). The gene displaying the highest degree of

sequence similarity to dystrophin is utrophin from ubiquitously expressed dystrophin

homologue or dystrophin-related protein 1 (DRP1), with around 51% amino acid

homology, similar exon/intron composition (Love et al., 1989, Pearce et al., 1993),

and smaller transcripts arising from internal promoters (Fabbrizio et al., 1995, Blake

et al., 1995). Smaller ancestral homologues include „torpedo‟ in electric rays (Carr et

al., 1989), „MSP-300‟ and „dah‟ proteins in fruit flies (Zhang et al., 1996), as well as

the „dystrobrevins‟ (Sadoulet-Puccio et al., 1996), „dystrotelins‟ (Jin et al., 2007) and

the vertebrate specific „DRP2‟ protein (Roberts et al., 1996) in humans and mice, all

of which retain significant similarity with the C-terminal and Cysteine-rich domains

of dystrophin.

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1.5 Animal models of DMD

The elucidation of dystrophin‟s evolutionary history has yet to facilitate the

elucidation of a precisely defined function for these proteins, but disruptive mutations

of early dystrophin homologues has provided useful information: Null mutations of

dys-1 protein in nematode worms result in subtle behavioural defects consisting of

hyperactive locomotion, exaggerated bending of the head, and a tendency to

hypercontract (Bessou et al., 1998), and in flies, MSP-300 null mutants have shown

that this protein contributes to muscle morphogenesis during development by

providing a functional link to laminin in the extracellular matrix, supporting the

integrity of the sarcolemma during or following myotubes extension (Rosenberg-

Hasson et al., 1996). This suggests a dual functional role for the single dystrophin

homologue found in invertebrates, as has recently been documented in C. elegans

(Zhou and Chen, 2011).

Utrophin has been show to be an autosomal paralogue of dystrophin, separated

by duplication at some point in the early evolution of vertebrates (Roberts and

Bobrow, 1998, Wang et al., 1998), and has received attention with regard to potential

therapeutic strategies aiming to replace dystrophin loss in DMD by up-regulating

utrophin expression. Indeed, utrophin expression has been found to be up-regulated

in both human DMD patients (Khurana et al., 1990) and in the murine „mdx‟ mouse

model of DMD (Love et al., 1991).

The mdx mouse arose as a spontaneous mutation in a mouse colony of

C57BL/10ScSn background (Bulfield et al., 1984) that results in premature

termination codon in the full-length dystrophin transcripts (Sicinski et al., 1989) and

is probably the most widely studied model of DMD due to its relatively low cost of

housing combined with short gestation and rapid maturation periods. Although the

limb muscle pathologies of humans and mice do differ, numerous similarities exist;

Delayed onset of disease pathology is observed in the mdx mouse, where muscle

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appears histologically normal until around one week of age and then between

approximately weeks 3-8 the muscles undergo cycles of degeneration and

regeneration with apparent synergistic interplay of the immune system and stem cell

populations (Turk et al., 2005). The acute degenerative period appears to end at

around 12 weeks of age, marked by the cessation of large scale immune cell

infiltrations and associated necrotic lesions within the tissue, thereafter muscles are

locked into a regenerative state. The condition changes once more, demonstrating

progressive fibrosis and muscle necrosis in animals of >1 year (Villalta et al., 2010).

Mdx animals do possess contractile and conductive deficits as well as cardiac dilation

and fibrosis that increase with age (Quinlan et al., 2004, Lefaucheur et al., 1995,

Pastoret and Sebille, 1995, Morrison et al., 2000), although the degree of fatty/fibrous

tissue formation in limb muscles is reduced in the mdx mouse compared with the

human pathology (Villalta et al., 2010). The mdx mouse does however, display

abnormal electrocardiogram readings and severely affected diaphragm by 6 months of

age (Bia et al., 1999), and it is the diaphragm muscle that is widely considered to be

the most reflective and reproducible of the human pathology (Stedman et al., 1991),

where dystrophy and fibrosis are both severe and progressive from an early age.

Feline and canine DMD models also exist. Cats display a pathology more reminiscent

of the mouse, and dogs that of humans (Khurana and Davies, 2003); hence golden

retriever muscular dystrophy (GRMD) (Cooper et al., 1988) may have addition

benefits for pre-clinical therapeutic assessment (Schatzberg et al., 1998).

1.6 Therapeutic approaches

Current therapeutic interventional approaches in DMD centre on efforts to re-

express or replace the functional elements of the dystrophin protein in muscle

(Amenta et al., 2010, Mendell et al., 2010). These therapies can be crudely grouped

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into gene-therapies, stem cell-therapies and pharmacological therapies. The latter are

also used in managing the condition. Gene-therapy based approaches seek to

artificially introduce DNA sequences into diseased tissues, with a view to achieving

tissue specific expression of normal or modified host-derived protein sequences able

to fulfil, or partially fulfil functions of proteins lost in the disease processes. Stem

cell based approaches share a similar rationale, seeking therapeutic gain through the

introduction of modified, host-derived stem cells or undifferentiated progenitor cells

to replace loss of function through their incorporation back into host tissues. Neither

avenue is without its problems, chief of which are the size of the gene to be replaced,

immunological rejection of foreign vectors/non-host derived cells, difficulties in

tissue specific delivery, lack of stable expression sufficient to confer functional gain

and possible toxicity/side effects.

Classical vector-borne gene therapy approaches for DMD were hindered by

problems of transgene size, which have been addressed using mini- and micro-

dystrophin cDNA constructs delivered most successfully via modified adenoviral in-

vivo and lentiviral vectors ex-vivo (Kochanek et al., 1996, Chen et al., 1997);

(Rodino-Klapac et al., 2011, Rodino-Klapac et al., 2010, Trollet et al., 2009).

Clinical trials research into scalable production techniques and immune tolerance of

these vectors remains ongoing in an effort to develop methods of supplying sufficient

amounts of packaged vector for full body application in humans.

Advances in modern molecular biology have facilitated the expansion of gene

therapy from the classical gene delivery-based systems into pharmacological based

attempts to repair/replace genetic defects in-situ. The well documented „reading

frame‟ hypothesis proposed by (Monaco et al., 1988) can explain ~90% of the

phenotypic differences observed between DMD/BMD patients (Prior and Bridgeman,

2005). The subsequent discovery of a DMD patient possessing a 52bp deletion in

exon 19, resulting in the transcriptional „skipping‟ of the entire exon 19 coding

sequence (Matsuo et al., 1991), led to the proposal of using targeted „exon skipping‟

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as a therapeutic approach for restoring the dystrophin reading frame and hence its

expression in DMD patients (van Deutekom et al., 2001). Exon skipping using

specific antisense oligonucleotides (AONs) has been applied in both the mdx mouse

and DMD patients, the therapeutic potential of local administration of such drugs has

been demonstrated in both mdx mice (Gebski et al., 2003) and DMD patients

(Aartsma-Rus et al., 2003) and clinical trials are currently ongoing (Kinali et al.,

2009); AVI Bio Parmaceutics plans phase II trials by the end of 2010 (AVI-4658) and

Prosensa/GSK are currently running phase III trials (PRO51). Another promising

pharmaceutical candidate has been PTC124, the aminoglycoside derivative known as

gentamycin, capable of inducing ribosomal read through of premature, but not native,

termination codons within the DNA sequence being transcribed (Welch et al., 2007,

Barton-Davis et al., 1999, Malik et al., 2010). This type of mutation would be

applicable to 10-15% of DMD cases. Phase II clinical trials using PTC124 (Ataluren;

PTC Therapeutics) demonstrated some beneficial effect; slowing the reduction in

average 6 minute walking distance over a 48 week period, although this effect was

dosage-dependent (Action Duchenne) and statistical analysis is ongoing. Antisense-

mediated exon skipping, together with forced stop codon read through currently

represent the most advanced clinical trials in the area of gene/gene-derived-therapies

for DMD (reviewed in (Aartsma-Rus et al., 2010, Sugita and Takeda, 2010, Guglieri

and Bushby, 2010).

Stem cell-based approaches have gained significant notoriety in the last

decade, paralleled by an increasing public awareness of their potential for major

therapeutic gains in a plethora of genetic disorders. Early endeavours exploited the

classical satellite cell population for subsequent myoblast re-implantation (Gussoni et

al., 1997, Fan et al., 1996) but subsequent studies revealed a plethora of cells types

and to date, more than a dozen different populations of myogenic precursor/facilitator

cells have been reported to offer potential therapeutic benefit for muscular dystrophy

patients including myoblasts, mesoangioblasts, pericytes, myoendothelial cells,

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CD133+ cells, aldehyde-dehydrogenase positive cells, mesenchymal stem cells,

embryonic stem cells, induced pluripotent stem cells and PICs (Price et al., 2007,

Quattrocelli et al., 2010, Meng et al., 2011, Tedesco et al., 2010, Negroni et al.,

2011). However, the dynamic nature of skeletal muscle represents a highly complex

arena in which to introduce proliferating cell types, many of which being multipotent.

Although stem cell based therapies do not share the capacity issues of viral or plasmid

derived vectors, there remain several obstacles to their efficient use in-vivo; Cellular

survival and host immune rejection have always been a consideration here

(Beauchamp et al., 1997, Mendell et al., 2010), as have satisfactory levels of

migration and transgene expression (Carnio et al., 2011, Skuk and Tremblay, 2003).

Yet, with so many potentially beneficial cell types coming to the fore, and cellular

delivery methods ever improving, the current „one cell to cure all‟ philosophy may

give way to cell therapies tailored to curing/prolonging the life of the individual and

most vital muscle groups.

While the search for effective gene- and cell-based therapies remains on going,

the currently available pharmacological interventions broadly aim to manage/improve

the phenotype for the patient. Corticosteroids such as prednisone and deflazacort

have long been used in DMD (Drachman et al., 1974, Barthelmai, 1965, Bonifati et

al., 2000, Muntoni et al., 2002) Although their exact mechanism of action remains

largely elusive and have been reported to be complex, they do confer limited benefits

in slowing disease progression., However, such benefits are often to some degree

offset by unwanted side effects such as osteoporosis, weight gain and cataracts (Fisher

et al., 2005). As muscle wasting is characteristic of the DMD phenotype, light

exercise programs are encouraged to delay these processes. The use of anabolic

steroids (Zeman et al., 1994) and creatine-based supplements have also been shown to

provide small improvements in muscle strength (Walter et al., 2000b).

The loss of muscle mass in DMD is commonly seen to represent a failure of

the organ to regenerate itself under sustained pathological degradation of its

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progenitor cell population through degradative exhaustion or premature aging

(Chamberlain, 2010). This has led to the exploration of possible solutions via growth

factor manipulation, where-by up-regulating muscle growth in situ may sufficiently

compensate for the degree of muscle loss seen in DMD to give some kind of

therapeutic benefit (McPherron and Lee, 1997); (Lee and McPherron, 2001).

Antibody-mediated myostatin inhibition has been shown to result in anatomical

improvement in the mdx mouse (Bogdanovich et al., 2002), and mdx mice with

myostatin mutations also display similar beneficial characteristics (Wagner et al.,

2002). Phase II clinical trials using a myostatin inhibitor (MYO-29, Wyeth

Pharmaceuticals) are still ongoing (Wagner et al., 2008). Attempts have been made to

enhance muscle mass in the dystrophic background by manipulating other growth

regulatory factors, such as insulin-like growth factor-1 (IGF-1) (Barton et al., 2002)

and follistatin (Iezzi et al., 2004). Furthermore, potential gene therapy- and gene

transactivation-based approaches, specifically through in situ manipulation of

utrophin promoter activity, are being exploited (functional parallels with dystrophin

underlying this approach have been discussed earlier). Should pharmacological

induction of utrophin expression prove successful, then such therapies could negate

the inherent difficulties of synthetic vector borne gene delivery (reviewed in Khurana

& Davies, 2003) and of immunogenicity of dystrophin protein introduced into

dystrophin-negative individuals. Unfortunately, so far BioMarin‟s 2010 phase I

clinical trial of compound BMN-195 has proved disappointing, with administrations

of up to 400mg/kg BMN-195 resulting in plasma concentrations below that believed

to be required for utrophin production (www.actionduchenne.org).

Calcium channel blockers such as nifedipine and diltiazam have also been

trialled as therapeutics (Khurana and Davies, 2003), but whereas increased

intracellular calcium concentration has been a recognised hallmark of the disease

since the work of (Bodensteiner and Engel, 1978) over 40 years ago, therapies aiming

at blocking Ca2+

channels have had limited success, so far (Jorgensen et al., 2011).

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The pathology of the disease does offer inviting openings for therapeutic

intervention; the delayed onset of the phenotype until around 5 years of age, the role

of the inflammatory response in muscle degeneration, the somatic induction of

revertant fibre expression in myonuclei (through restoration of the dystrophin reading

frame) (Hoffman et al., 1990, Nicholson et al., 1989), and perhaps most intriguingly,

the documented absence of disease pathology in smaller muscle groups such as the

extraocular muscles (EOM) (Karpati and Carpenter, 1986), a phenomenon recently

reported to relate to the more capable calcium handling properties of the EOM

compared with TA muscle (Zeiger et al., 2010), highlighting a resurgence in efforts to

elucidate the role of Ca2+

in dystrophic muscle.

1.7 Calcium signalling and DMD

The notion of Ca2+

acting as a second messenger in signal transduction is long

established (Rasmussen et al., 1990, Ringer, 1883), and since much of the early

studies were conducted in cardiac and skeletal muscle (reviewed in (Ebashi, 1991), a

large body of work already existed for those seeking to explain the abnormalities in

Ca2+

-mediated signalling so often reported to coincide with the degenerative

pathology in DMD patients. Abnormalities in Ca2+

homeostasis have long been

postulated as effectors of the dystrophic pathology in humans and animals (Bertorini

et al., 1982b, Bertorini et al., 1982a, Turner et al., 1988, Hopf et al., 2007, Gailly,

2002, Batchelor and Winder, 2006, Jorgensen et al., 2011, Allen et al., 2010a, Morine

et al., 2010, Zeiger et al., 2010), yet whether the mechanisms by which dystrophin

loss results in abnormally high intracellular Ca2+

levels in dystrophic muscle have

been fully elucidated remains the topic of much debate (reviewed in Allen et al, 2010;

Batchelor & Winder, 2006; Hopf et al, 2007). The hypothesis relating to the

dystrophin protein functioning as a shock absorber, tethering its associated protein

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complex to distinct areas of the muscle membrane has been extended in relation to the

DMD pathology, with the suggestion that loss of dystrophin, aside from resulting in

loss of the functional link between the actin-cytoskeleton and laminin in the

extracellular matrix, may actually result in contraction-induced damage and

deregulation of mechanosensitive Ca2+

channels as the internal myosin machinery is

left to move freely inside the sarcolemma. The notion of calcium abnormalities in

dystrophic muscle was actually suggested prior to the identification of the DMD gene

(Bertorini et al., 1982a, Bertorini et al., 1982b) and several routes of entry for the

observed increased levels of intramuscular calcium were proposed; with increases in

sarcolemmal fragility reported in both DMD patients and mdx mice (Petrof et al.,

1993, Watkins et al., 1988, Beam, 1988). Another opinion has evolved that, rather

than contraction-induced necrosis of entire myofibres, the degree of survivable

sarcolemmal disruptions is up-regulated in the dystrophic pathology (Alderton and

Steinhardt, 2000b, Alderton and Steinhardt, 2000a), where increases in survival have

been documented following reductions in extracellular calcium levels (Gailly, 2002).

This hypothesis has itself evolved as other mechanisms for Ca2+

entry into dystrophic

muscle have been proposed; micro membrane ruptures, aberrant activation of

sarcoplasmic reticulum (SR) calcium channels, sarcolemmal leak channels and Ca2+

buffering mechanisms (reviewed in (Batchelor and Winder, 2006, Gailly, 2002, Allen

et al., 2005), leading to the proposition that fast and localised changes in membrane

integrity, permeability and therefore intracellular Ca2+

concentrations ([Ca2+

]i) give

rise to unscheduled Ca2+

sparks within muscle fibres (reviewed in (Niggli, 1999,

Batchelor and Winder, 2006). Regardless of the route of entry, most researchers

agree that the intracellular accumulation of Ca2+

can be toxic to all living cells and its

role in the activation of cellular proteases and the apoptotic cascade has been well

documented in normal and diseased muscle (Alderton and Steinhardt, 2000a, Mallouk

et al., 2000). However, the suggestion has also been made that the increased [Ca2+

]i

in dystrophic muscles may actually be beneficial and involved in repairing the

20

damage to membrane ruptures via stress-induced membrane repair involving dysferlin

and annexin recruitment to sites of membrane ruptures (Bansal et al., 2003,

Lammerding and Lee, 2007). With a recent revival in interest surrounding Ca2+

signalling in DMD, possibly owing to the lull in new ideas surrounding the structural

role of dystrophin, the search for a unifying hypothesis to explain the pathology has

given way to a more combined rationale, where dystrophin is seen as a much more

multifunctional protein able to combine interlinked faculties including dystrophin-

associated complex retention, structural definition and support, signalling cascade

governance and cellular fate determination. In studying the potential roles for Ca2+

in

the dystrophic pathology, researchers have focussed largely on the detrimental effects

of increases in [Ca2+

]i, largely due to the pre-existing and well documented role of

SR-derived Ca2+

release in malignant hyperthermia in muscle (Lopez et al., 1985,

Blanck and Gruener, 1983). More recent studies started to unravel potential

mechanisms by which Ca2+

sparks could induce not only the apoptotic and necrotic

signalling cascades, but also those resulting in growth, proliferation, differentiation

and cellular survival (reviewed by (Batchelor and Winder, 2006).

21

Figure 1.7. Calcium abnormalities in DMD muscle. Illustration depicting possible origins

of calcium signalling perturbations in dystrophic muscle resulting from loss of Dp427

expression (red). Dystrophin mutations result in loss of connectivity between extracellular

matrix and cytoskeletal filaments (green) through DAPC complex members at the

sarcolemmal membrane. Associated abnormalities in calcium homeostasis result in

disruption of normal intracellular signalling cascades, with complex downstream

consequences.

Elucidating the molecular mechanisms of Ca2+

signalling in a complex and

plastic tissue such as muscle, is difficult. Adding to that the consideration that

dystrophic muscle endures sustained and often highly localised cycles of degeneration

and repair often associated with relentless host immune cell invasion, it becomes plain

to see why the intricacies of Ca2+

role in DMD remain elusive. Yet there is one area

of molecular biology that is well accustomed to such considerations; that of tumour

biology. The life or death paradox surrounding Ca2+

signalling in cancer may lend

22

itself well to that of muscular dystrophy (reviewed by (Monteith et al., 2007), more

specifically the area of purinergic calcium channels could be of particular interest in

the area of muscle diseases, where high ATPe concentrations abound.

1.8 Purinergic signalling and receptors

Although purinergic responses were originally characterised using adenine

compounds (Bennet and Drury, 1931, Drury and Szent-Gyorgyi, 1929) to stimulate

the heart, the signalling role of the related compound, ATP, was not fully appreciated

until much later. Geoffrey Burnstock is credited with having coined the term

„purinergic transmission‟ to describe the actions of ATP on the gut and bladder

(Burnstock, 1972). It was he who postulated that membrane receptors for ATP must

exist due to the universal nature of its action on multiple cell types (Burnstock, 1976),

and later went on to describe the basis for distinguishing two separate families of

ATP receptors; the cation channels and the G-protein coupled receptors. Both types

are membrane bound and belong to large families of structurally and

pharmacologically related ATP receptors. The currently accepted nomenclature

describing purine receptors includes: P1 (A1,A2A, A2B, A3) and P2 (X and Y) (see

(Burnstock, 2007), for a review). All P1 receptors are membrane bound G-protein

coupled receptors, mostly acting via the activation or inhibition of adenylate cyclase.

Recognising adenosine as their principal agonist, P1 receptors share 39-61% sequence

homology with each other and 11-18% with the P2Y receptors.

P2Y receptors share structural characteristics within the family and similarities

with the P1 receptor subtype. They are also G-protein-linked, with seven

transmembrane domains, an extracellular N-terminus and intracellular C-terminus but

differ in agonist selectivities. Collectively, P2Y receptors recognise a host of purine

and pyrimidine agonists, such as ATP, ADP, UTP and UDP, typically in the sub-

23

micromolar range, and signalling is generally conducted via IP3 and cAMP induced

mobilisation of intracellular Ca2+

. Eight human P2Y receptors have been cloned to

date (P2Y1, 2, 4, 6, 11, 12, 13 and 14) (Burnstock, 2007), differentiated by their

unique pharmacological profiles in response to various concentrations of ATP, ADP,

AMP, UTP, and UDP and synthetic agonists and antagonists.

P2X receptors are structurally distinct from the P1 and P2Y subtypes

(Burnstock and Kennedy, 1985), forming non-selective cation channels consisting of

homo and hetero-subunit trimers. Individual subunits comprise intracellular N and C-

termini that serve as binding motifs in protein kinase mediated signalling, two

membrane spanning domains, which collectively form the membrane channel of the

trimer, and a large extracellular loop containing the ATP binding pocket orientated

by a structurally conserved disulphide-bridge domain of 10 cysteine residues (North,

2002, Nicke et al., 1998). The pharmacological profiles of P2X receptors also set

them apart from other related families, the principal agonist here being ATP, which

binds all P2X receptors with varying affinities in the low M to mid mM range.

Seven P2X receptor subunits (P2X1-7) have been cloned and characterised, sharing

30-50% sequence homology at the peptide level (North, 2002). Collectively, this

family of nucleotide receptors represents a staggeringly complex yet elegantly

intricate matrix of signal transduction, conferring to the individual cell the power to

exert exacting refinements in a myriad of metabolically driven reactions, and to

achieve this in paracrine, endocrine and exocrine mode/fashion. Many of these

receptors have become tissue specific through the course of evolution. The diversity

of function within this receptor family is beyond the scope of this study,

comprehensive reviews include Burnstock & King, 1996; Burnstock, 2007, (White

and Burnstock, 2006, Ralevic and Burnstock, 1998, Burnstock et al., 2010,

Surprenant and North, 2009).

24

Figure 1.8. Predicted architectures of purinergic receptor subtypes. Illustration

depicting the main structural and functional features of purinergic receptors: P1 and P2Y

receptors are G-protein coupled receptors comprising seven membrane spanning domains,

intracellular C termini and extracellular N termini (A). P2X receptors comprise two

transmembrane domains, with intracellular N and C termini (B). P2X receptors classically

form hetero- and homo-trimeric, non-selective ion channels gated by amongst others, ATPe

[C; closed (left) and open (right); three predicted ATP binding sites are indicated (dark blue

crescents)]. P2X7 receptors are unique amongst other P2X receptors, possessing an extra

long C-terminal tail, and also possess 10 conserved extracellular cysteine residues (red

circles). Schematic also illustrates some functional implications of mutagenesis studies using

P2X7 receptors expressed in HEK-293 cells (D).

1.9 Purinergic systems in DMD

Of the P2 receptors that may be of interest with regard to neuromuscular

disorders, the P2X family stand out as the most likely to show pronounced alterations

in response under disease conditions, due to its heightened specificity for the one

principal agonist, ATP. ATP has long been known to exist at high concentrations (5-

10mM) within skeletal muscle (Carlson and Siger, 1960, Walter et al., 2000a).

25

Moreover, it has been shown to be released into the extracellular milieu of multiple

tissue types via lytic and non-lytic mechanisms. These were most extensively studied

in epithelial and endothelial cells following shearing stresses, compression, stretching,

hydrostatic pressure, hypotonic shock and necrotic cell death (Grierson and

Meldolesi, 1995, Sauer et al., 2000, Ferguson et al., 1997, Hazama et al., 1999,

Grygorczyk and Hanrahan, 1997, Idzko et al., 2007, Martinon, 2008). Such studies

have shown extracellular ATP (ATPe) release to reach concentrations of up to

500M, which would be sufficient to activate all P2X receptor subtypes. However,

there is some on-going debate as to whether the concentrations of extracellular ATP,

which actually occur in-vivo are of sufficient level and duration to overcome the

activity of nucleotide- metabolising enzymes (ecto-nucleotidases) located on the outer

surface of the membrane - It has been suggested that at concentrations of >100μM,

ecto-nucleotidases cannot degrade sufficient amounts of ATP, leading to a sustained

high level of extracellular ATP (Bodin and Burnstock, 1996) resulting in tissue

damage (Neary et al., 1994). It seems less likely that ecto-nucleotidases would be

sufficient to entirely abate the effects of substantial cellular ATP release from skeletal

muscles, especially in the case of the dystrophic pathology, where dystrophin absence

results in the loss of -sarcoglycan from the sarcolemma, which is itself an ecto-

ATPase enzyme (Betto et al., 1999, Sandona et al., 2004).

The expression of P2X2, 4, 5, and 7 receptors has been independently

documented in muscle development (Meyer et al., 1999, Ryten et al., 2001), muscle

regeneration following dystrophic degeneration (Ryten et al., 2004, Yeung et al.,

2004), and in myoblast lines (Ryten et al., 2002, Yeung et al., 2006). Of these, only

P2X5 has been assigned any prospective function, with a proposed involvement in the

differentiation of myoblasts to myotubes but this was based only on differences in

antibody staining intensities (Ryten et al., 2002, Ryten et al., 2004). Furthermore,

considering the hive of interest surrounding the elucidation of the molecular

26

mechanisms pertaining to myogenic precursor lineage and fate determination, it is

surprising to note that this work was never followed up in the literature.

Although the concentrations of ATP found within normal skeletal muscles are

more than sufficient to activate all P2 receptors, one receptor in particular, P2X7,

stands out as a clear candidate for generating the most observable changes in diseased

muscle. P2X7 is the least sensitive to its principal agonist (ATP) of all the P2

receptors, its EC50 being >300M, which is about 100 fold higher than other P2X

receptors (North, 2002). It is also distinguished from other family members by its

pore-forming ability, which has been ascribed to its long (240 amino acids),

cytoplasmic C-terminal (Surprenant et al., 1996). While all P2X receptors have been

implicated in many diverse processes; growth, proliferation, migration, apoptosis,

cancer, epithelial transport, cytokine release, apoptosis and cancer (Ralevic and

Burnstock, 1998) the orthodox opinion of P2X7 describes it as a „death-inducing

receptor‟. It is because in some cells, prolonged P2X7 stimulation leads to the

formation of a lytic pore, in addition to the ion channel function described above. This

pore, first characterised in rat mast cells (Cockcroft and Gomperts, 1980, Cockcroft

and Gomperts, 1979), facilitates the permeation of large molecules (<900 Daltons)

through the plasma membrane, as demonstrated using various dyes, such as ethidium

bromide, lucifer yellow, and YOPRO-1. This permeation pathway can induce

cytoskeletal rearrangements of actin and tubulin, translocation of phosphatidyl serine

to the outer membrane leaflet, membrane „blebbing‟ and microvesicle shedding, as

well as changes in mitochondrial size, shape and efficiency (for reviews, see (North,

2002, Ralevic and Burnstock, 1998, Burnstock and Knight, 2004). Investigations into

the mechanisms of pore formation in macrophages and transfected HEK cells have led

to two potentially distinct mechanisms being proposed: firstly that P2X7 subunits are

recruited in greater numbers following prolonged ATP stimulation, thus widening the

pore, allowing for the passage of larger molecules (Di Virgilio et al., 1999).

Secondly, that the covalent recruitment of another protein or complex of proteins

27

occurs, which creates the membrane pore when triggered to do so by P2X7 (Pelegrin

and Surprenant, 2006). Here, the best documented candidate is the pannexin-1 hemi-

channel (Pelegrin and Surprenant, 2006), although this could not be confirmed in cells

from pannexin-1 knockout animals (James Elliot, personal communication). Neither

hypothesis has been universally accepted or rejected (Yan et al., 2008). Moreover, it

has been reported that the small cation channel pathway can be differentiated from the

NMDG/YO-PRO permeability pathway, but that the NMDG and YO-PRO uptake

mechanisms can themselves be different by some as yet undefined mechanism.

More recently, attention has shifted from the P2X7 lytic pore forming

properties to the well known, yet comparatively little studied, ability of this receptor

to control fast cation fluxes at the cell membrane, which serve to initiate a wealth of

signalling cascades. It has become apparent that lytic pore formation is not a

requirement for the induction of apoptosis/necrosis in P2X7-expressing cells; it has

been shown that pore formation and apoptotic induction following P2X7 activation

are indeed often reversible. Hence researchers have tentatively coined terms such as

„pseudo-apoptosis‟ (Mackenzie et al., 2005) and „aponecrosis‟ (Elliott and Higgins,

2004) to describe these phenomena; the accuracy, relevance and true mechanism of

which are still a mystery and often cell/tissue specific.

There has been an increase in attempts to characterise the intricate mechanics of

intracellular P2X7-mediated signal transduction. The main focus has been on the

conditions surrounding pathological/disease states, such as neurological/psychiatric

disorders, cardiovascular disease, inflammatory diseases and cancer, as it is now

widely accepted that conditions such as stress, trauma and inflammation can lead to

the release of high concentrations of ATP form damaged tissues (for comprehensive

reviews, see (Skaper et al., 2009, Di Virgilio et al., 2009). From these studies, an

intriguingly complex, time-and concentration-dependent system of ATP release and

signalling is emerging now. It appears that low level or tonic stimulation at low ATP

concentrations (mid M range) of P2X7 receptors may be beneficial to the cell

28

through stimulation of its growth, proliferation, metabolic, or migratory pathways.

The most well documented are effects mediated by alterations to the Ras, Raf, MEK,

ERK, Fos/Jun mitogen activated protein kinase (MAPK) pathway. Interestingly, the

dependence of this kinase pathway on calcium influx has been shown to be cell type

specific (Amstrup and Novak, 2003). As stimulation progresses from the low level,

tonic to the sustained high concentration (low mM range), the cellular response then

shifts to being detrimental and even excitotoxic. This phase can also be divided by its

calcium dependence: the calcium mediates phosphatidyl serine translocation,

membrane blebbing, microvesicle shedding, cytoskeletal disruptions, all of which

seem reversible upon removal of stimulus, and the calcium dependent, caspase-3-

mediated apoptotic pathway. The mechanisms of how this Ca2+

dependence is

transmitted and whether this division can be uniformly applied to multiple cell types

remain unknown.

The paradoxical nature of P2X7 up-regulation in tumours may potentially

serve to provide analogies to the purinergic phenotype observed in dystrophic muscle

cells. However, it is puzzling to consider that a tumour may overexpress a receptor so

sensitive to excitotoxicity in an environment so rich in its stimulating agonist. This

observation has led researchers to adjust their hypotheses on P2X7 roles in disease,

and, in doing so, to provide what may become an interdisciplinary view of signalling

integration. This view, when applied to DMD, may go some way to explaining the

complex degenerative/regenerative stimuli underlying the dystrophic pathology, with

a view to future therapeutic intervention. Abnormalities in ATPe responses in

dystrophic myoblasts have been documented previously (Yeung et al., 2006),

necessitating the more in-depth investigations into the mechanism of this response

that are described in this study.

29

1.10 Research hypothesis

Changes in P2X7 receptor function contribute to the pathology of DMD.

1.11 Thesis aims

Characterise the pharmacological ecto-ATP hydrolysing profiles of normal and

dystrophic myoblasts and myotubes and thus evaluate the purinergic

significance of -sarcoglycan loss from the cell membrane of dystrophic cells.

Investigate P2X7 expression in normal and dystrophic muscle cell cultures in

vitro and muscles in situ; characterise mRNA levels, splice variants expression

as well as protein profiles and localizations.

Analyse functional differences in P2X7 responses between wild-type- and

dystrophic- derived muscle cell cultures.

Characterise using mass spectrometry the immediate proteome response of

normal and dystrophic myoblasts following exposure to extracellular ATP.

Determine the effect of P2X7 receptor blockade in the mdx mouse in vivo

through pharmacological inhibition.

30

2

Materials & Methods

2.1 Chemicals and reagents

Table 2.1. Chemical and reagents used in this study

Product name (alphabetical) Supplier

A

Acetic acid, glacial Fisher Scientific Ltd.

Acrylamide/bis Bio-Rad Laboratories

Agar Sigma-Aldrich Ltd.

Agarose Biogene Ltd.

Alexa Fluor 563 donkey anti-goat IgG Invitrogen Ltd.

Alexa Fluor 488 chicken anti-rabbit IgG Invitrogen Ltd.

Alexa Fluor 488 goat anti-rabbit IgG Invitrogen Ltd.

Ammonium Persulphate Sigma-Aldrich Ltd.

Ampicillin Sigma-Aldrich Ltd.

Anti--actin antibody Sigma-Aldrich Ltd.

Anti-collagen IV antibody Millipore

Anti-dystrophin antibody (2166) Gift from Collaborators

Anti-dystrophin antibody (7A10) Developmental Studies Hybridoma

Bank (DSHB)

Anti-ERK 1/2 antibody Cell Signalling Ltd.

Anti-F4/80 antibody Abcam

Anti-myf-5 antibody Santa Cruz Ltd.

Anti-NG2 antibody Millipore

Anti-pannexin-1 antibody Santa Cruz Ltd.

Anti-phospho-ERK 1/2 antibody Cell Signalling Ltd.

Anti-P2X7 antibody Cell Signalling Ltd.

Anti-goat IgG, biotin-labelled Vector Laboratories Ltd.

Anti-goat IgG, peroxidase-labelled Sigma-Aldrich Ltd.

Anti-mouse IgG, biotin-labelled Sigma-Aldrich Ltd.

31

Anti-rabbit IgG, biotin-labelled Vector Laboratories Ltd.

Anti-rabbit IgG, peroxidase-labelled Sigma-Aldrich Ltd.

Adenosine triphosphate disodium salt Sigma-Aldrich Ltd.

Avidin biotin blocking kit Vector Laboratories Ltd.

B

Bicinchoninic acid kit Sigma-Aldrich Ltd.

Betadine Gift from Marta Onopiuk

Bovine serum albumin Sigma-Aldrich Ltd.

β-mercaptoethanol Sigma-Aldrich Ltd.

BzATP (2'(3')-O-(4-Benzoylbenzoyl)adenosine-

5'-triphosphate tri(triethylammonium) salt) Sigma-Aldrich Ltd.

C

Calcium chloride Sigma-Aldrich Ltd.

Chick embryo extract Sera Labs Ltd.

Collagenase, type 1 Sigma-Aldrich Ltd.

Coomassie brilliant blue G Sigma-Aldrich Ltd.

Cryo embedding medium Raymond A Lamb Ltd.

Cysteine Sigma-Aldrich Ltd.

D

dATP Invitrogen Ltd.

dCTP Invitrogen Ltd.

dGTP Invitrogen Ltd.

dTTP Invitrogen Ltd.

Distilled water, RNase DNase free Invitrogen Ltd.

Dithiothreitol Sigma-Aldrich Ltd.

DMEM Lonza Ltd.

DNA ladder; 100 bp Invitrogen Ltd.

DNA ladder; 1 kb Invitrogen Ltd.

DNase I; amplification grade Invitrogen Ltd.

Donor horse serum Sera Labs Ltd.

DPX mounting medium Fisher Scientific Ltd.

E

96% EtOH Fisher Scientific Ltd.

100% EtOH Fisher Scientific Ltd.

ECL Western blotting detection system Cheshire Biosciences Ltd.

32

EcoRI New England Biolabs Ltd.

Ethylenediaminetetraacetic acid (EDTA) Sigma-Aldrich Ltd.

Ethylene glycol tetraacetic acid (EGTA) Sigma-Aldrich Ltd.

Ethidium bromide solution Sigma-Aldrich Ltd.

F

Faramount® aqueous mounting medium Dako Cytomation Ltd.

Foetal bovine serum (FBS) Lonza Ltd.

Formaldehyde Fluka Biochemika

G

Glycerol Sigma-Aldrich Ltd.

Glycine for electrophoresis

GoTaq polymerase

Sigma-Aldrich Ltd.

Promega Ltd.

H

Haematoxylin and eosin kit Sigma-Aldrich Ltd.

HEPES Sigma-Aldrich Ltd.

Hind III New England Biolabs Ltd.

Hybond P membrane Amersham Pharmacia Biotech

Hydrogen peroxide Sigma-Aldrich Ltd.

I

Isopropyl-beta-D-thiogalactopyranoside (IPTG) Invitrogen Ltd.

Isopentane Sigma-Aldrich Ltd.

Isopropanol Fluka Biochemika

J

JM109 competent cell tranfection kit Promega Ltd.

L

Laemelli sample buffer Bio-Rad Laboratories Ltd.

LB broth Sigma-Aldrich Ltd.

M

Magnesium chloride BDH Chemicals Ltd.

Methanol Fisher Scientific Ltd.

Methyl green

Mini elute gel extraction kit

Vector Laboratories Ltd.

Qiagen Ltd.

33

N

N,N,N′,N′-tetramethylethylenediamine (TEMED) Sigma-Aldrich Ltd.

Normal chicken serum Vector Laboratories Ltd.

Normal goat serum Vector Laboratories Ltd.

Normal horse serum Vector Laboratories Ltd.

Normal rabbit serum Vector Laboratories Ltd.

NP-40 Sigma-Aldrich Ltd.

Nuclease free water Fisher Scientific Ltd.

P

pGEM-T easy vector kit Promega Ltd.

Paraformaldehyde Sigma-Aldrich Ltd.

Penicillin Sigma-Aldrich Ltd / Lonza Ltd.

Phosphate buffered saline tablets Sigma-Aldrich Ltd.

PCR master mix PromegaLtd.

Phenol:chloroform:isoamyl alcohol (47.5:47.5:1) Sigma-Aldrich Ltd.

Poly-L-lysine Sigma-Aldrich Ltd.

Potassium chloride Fisher Scientific Ltd.

Protease inhibitor cocktail tablet; complete mini Roche Diagnostics Ltd.

R

Ready gel 10% tris-glycine precast gels Bio-Rad Laboratories Ltd.

RNaseOut Invitrogen Ltd.

RNeasy minikit (50) Qiagen Ltd.

S

Sodium acetate Sigma-Aldrich Ltd.

Sodium azide Sigma-Aldrich Ltd.

Sodium borohydride Fisher Scientific Ltd.

Sodium chloride Sigma-Aldrich Ltd.

Sodium dodecyl Sulphate Sigma-Aldrich Ltd.

Sodium fluoride Sigma-Aldrich Ltd.

Sodium hydroxide Sigma-Aldrich Ltd.

Sodium phosphate, dibasic Sigma-Aldrich Ltd.

Sodium phosphate, monobasic Sigma-Aldrich Ltd.

Sodium orthovanadate Sigma-Aldrich Ltd.

Sucrose Sigma-Aldrich Ltd.

SuperScript III reverse transcriptase Invitrogen Ltd.

34

T

T4 DNA ligase

Taq DNA polymerase

Promega Ltd.

Promega Ltd.

Triton X-100 Sigma-Aldrich Ltd.

Trizma® base Sigma-Aldrich Ltd.

Trizma® hydrochloride Sigma-Aldrich Ltd.

Trizol Invitrogen Ltd.

Trypsin-EDTA (10X) Lonza Ltd.

Tween-20 Sigma-Aldrich Ltd.

V

VectaShield® mounting medium Vector Laboratories Ltd.

VectaStain® ABC-AP kit Vector Laboratories Ltd.

Vector® VIP Substrate kit Vector Laboratories Ltd.

X

X-Gal Promega Ltd.

Xylene Fisher Scientific Ltd.

35

2.2 Animals

All research animals were supplied by Portsmouth University Bioresources

Centre, where all animal procedures were performed in accordance with the Local

Ethical Review Committee agreements and UK Home Office Animals (Scientific

Procedures) Act 1986.

2.2.1 Mice

Mice were bred and maintained under an environment conditioned at 19-

23oC (with humidity at 45-65%) with 12 hr light/dark cycle. They were fed a

standard pellet diet (economy rodent breeder diet, special diet services, Witham,

UK) and tap water ad libitum. The animal beddings (Goldmix) provided were

further enriched with shredded papers and sunflower seeds.

C57BL10 & mdx strains: mdx and C57BL10 mice were used, the latter

as the normal background to the mdx geneotype. All strains are tested routinely

for main pathogens and all were healthy.

2.2.2 In vivo injections:

C57BL10 and mdx male mice were used in accordance with approvals of

the institutional Ethical Review Board and the UK Home Office. Mice (3-9 per

group) received 125mg/kg b.w. Coomassie Brilliant Blue G 250 (Sigma)

intraperitonealy (i.p.), in sterile saline, according to two dosage regimes: daily

from weeks 3-6 and every three days from weeks 3 to 14. Control mice

received the same volume of saline solution. At 16 weeks mice were killed and

36

organs immediately flash frozen in isopentane cooled in liquid N2. Injections

were carried out by Prof. Gorecki, dissections and sample preservations were a

combined effort between Prof. Gorecki and the candidate.

2.3 Cell Culture

2.3.1 Immortalised H2Kb-tsA58 and H2K

b-tsA58/mdx myoblast lines

The Immortalized H2Kb-tsA58 and H2K

b-tsA58/mdx myoblast cell lines

were a gift from Prof. Hans Lochmüller (University of Newcastle). Culture

media for both lines comprised Dulbecco's modified Eagle's medium (DMEM)

supplemented with 20% (v/v) foetal bovine serum (FBS), 2mM L-glutamine,

100 U/ml penicillin, 100ug/ml streptomycin and 20 unit/ml murine -interferon.

Cells were grown in T25 flaks (Greiner) and passaged using 1X Trypsin (Lonza)

when approaching 70-80% confluency. Incubation conditions consisted of a

humidified atmosphere (95% air, 5% CO2) at 33C for myoblast proliferation,

switching to 37C, with removal of -interferon and substitution of 20% (v/v)

FBS for 10% (v/v) horse serum (HS) to induce myoblast to myotube

differentiation.

2.3.2 C2C12 Immortalized myoblasts

C2C12 mouse myoblast cells purchased from the European Collection of

Cell Culture (ECCC; #91031101) and were cultured in a medium comprising

DMEM supplemented with 20% (v/v) FBS, 2mM L-glutamine, 100 unit/ml

37

penicillin and 100ug/ml streptomycin. Incubation conditions consisted of a

humidified atmosphere (95% air, 5% CO2) at 33C for myoblast proliferation,

switching to 37C and substituting 10% (v/v) FBS for 10% (v/v) HS to induce

myoblast to myotube differentiation.

2.3.3 C57BL10-/mdx-derived primary myoblast cultures

First generation primary myoblasts: Freshly dissected hind limb

muscle groups [tibialis anterior (TA), gastrocnemius (GC), soleus (Sol), and

flexor digitorum brevis (FDB)] from male 4 month old C57BL10 and mdx mice

were used to establish primary myoblast cultures as described by (Rosenblatt et

al., 1995a). Briefly, muscles were dissected tendon to tendon where possible

with great care taken to avoid stretching or compression induced damage.

Dissected muscles were washed in 5ml 1X Hank‟s Buffered Saline Solution

(HBSS) containing 2 drops of Betadine (100mg/ml povidone-iodine) to sterilise

the tissues and remove bacterial contamination from the surface of the skin.

Sterilised muscles were then incubated in 0.1% (w/v) type 1 collagenase in a

shaking water bath at low speed for 45 mins. Muscles were then transferred to a

6 well plate (Greiner) containing 5ml plating medium (DMEM supplemented

with 10% HS, 0.5% chick embryo extract (CEE) and 2mM L-glutamine) using a

25ml pipette. Individual fibres were then dissociated through gentle pipetting

with reducing diameter pipettes (25, 10 and 1 ml). Isolated single living muscle

fibres were selected under a bright field microscope (Leica) and transferred to

5ml of fresh plating medium three times to wash the fibres and remove

contaminating cell types prior to plating. Single fibres (in 20ul plating media)

were plated in 24 well plates (Greiner) pre-coated with 100ul 1mg/ml Matrigel

and left for 5 mins to adhere before the addition of a further 1ml plating media.

After 3-5 days, mono-nucleate cells could be seen migrating off the fibres. At

38

around 7 days, medium was changed to proliferation medium (DMEM

supplemented with 20% FBS, 5% CEE and 2mM L-glutamine) and cultures

were observed to proliferate.

Second generation primary myoblasts: Due to the tendency of the

above protocol to induce rapid differentiation of isolated myoblast cultures, a

second primary isolation protocol was developed. This 2nd

generation protocol

used the same method of isolating living myofibres as described above, but

differed in the method of mononuclear cell culture establishment. The 2nd

generation myoblasts were cultured using novel growth media [20% Knockout

Serum Replacement (KRS; Invitrogen), 10% Donor Horse Serum, sterile filtered

(DHS; Sera Labs), 2mM L-glutamine]. This media was used throughout,

„plating‟ media was completely omitted from the 2nd

generation procedure.

KRS media was observed to greatly reduce the tendency of isolated myoblasts

to differentiate; myogenic cells retained a more spheroidal appearance and

proved easy to separate from any contaminating non-myogenic cells, which

adhered strongly and adopted a more elongated constitution. In contrast to the

1st generation procedure, myofibres isolated using the 2

nd generation procedure

did not adhere to the Matrigel substratum. This effect was thought to be due to

differences in media composition. Indeed, the lack of adherent fibres proved

crucial for the isolation of pure myoblast cultures; clonal colonies of both

myogenic and non-myogenic cells were obtained from cleanly isolated

myofibres, which deposited single cells randomly across the Matrigel

substratum if plates were gently agitated twice a day for 2-3 days. This

facilitated the subsequent purification of myoblast colonies from colonies of

contaminating cell types, which actually had a strongly differentiation inducing

influence over proximal myoblast or mixed myoblast/contaminating cell

colonies. After 2-3 days or once 5-6 colonies of cells were observed per 3.5cm

plate, myofibres were removed. Myoblast colonies were purified and separated

39

from contaminating cell types using a modified pre-plating technique: media

was removed and cultures were washed with 1ml 1X Cell Dissociation Solution

(CDS; Sigma) for 1 min. CDS was removed and the plates were gently tapped 5

times in the horizontal plane to induce cellular detachment. 2ml of fresh media

was added and the isolated cells re-plated in fresh Matrigel-coated plates (100ul

2mg/ml Matrigel / 3.5cm plate; Greiner). This pre-plating procedure was

repeated 3 times over the first week of culture to ensure purely myogenic

cultures had been derived. Using this technique, pure myogenic cultures could

be reproducibly obtained, based on their ability to form 100% contractile

myotubes.

2.3.4 Human embryonic kidney 293 (HEK) cells

HEK cells and HEK cells transfected with P2X7 were a gift from Dr.

Friedrich Koch-Nolte, Hamburg, Germany. Cells were cultured in a medium

comprising DMEM supplemented with 10% (v/v) FBS, 2mM L-glutamine, 100

unit/ml penicillin and 100ug/ml streptomycin in a humidified atmosphere (95%

air, 5% CO2) at 37C.

2.4 Molecular Biology

2.4.1 Total RNA extraction

Total ribonucleic acids were extracted from cultured cells according to

the manufacturer‟s instructions using Trizol (Invitrogen). Briefly, 50-100mg of

tissue was weighed and added frozen to 1ml Trizol and crushed completely

40

using 20 passes of a 10ml Potter-Elvehjem homogeniser (Fisher). Cellular

debris was removed through centrifugation at 5,900g for 10 mins at 4C in 1.5ml

Eppendorf tubes. Supernatants were transferred to new tubes containing 200l

Chloroform and shaken vigorously for 15s then incubated at RT for 2-3 mins.

Samples were centrifuged at 5,900g for 15 mins at 4C to separate phases. The

upper aqueous phase containing RNA was decanted into a fresh Eppendorf tube

and RNA precipitated through addition of 0.5ml isopropanol followed by

several inversions and incubation at RT for 10 mins. RNA was pelleted through

centrifugation at 5,900g for 10 mins at 4C. Pellets were washed in 1ml ice-cold

75% nuclease-free EtOH, re-pelleted through centrifugation at 3,300g for 5 mins

at 4C, then air dried for 5 mins and re-suspended in 20-30l nuclease-free

water.

2.4.2 Determination of RNA/DNA concentration

Small aliquots of total RNA/DNA samples were diluted 1:800 with

ddH2O and their absorbances measured at wavelength of 260 nm and 280 nm in

quartz cuvettes (Sigma-Aldrich Ltd) using a BioMate 3 Series

spectrophotometer (Thermo Electron Corporation Ltd). Total RNA

concentration was determined after taking into account the absorbance at 260

nm, the dilution factor, as well as the A260

value of 1 corresponding to 40 g/ml

(i.e. 50 g/ml for DNA; see below). Purity of RNA could be ascertained based

on the ratio of absorbances at 260 nm and 280 nm (i.e. A260

/A280

).

41

2.4.3 cDNA synthesis

Complementary DNA (cDNA) was synthesised from total RNA

according to manufacturer‟s instructions using Superscript III reverse

transcriptase (Invitrogen). Briefly, 2g of RNA was incubated for 25 mins at

25C in a 10l reaction buffer containing 200 mmol/l Tris-HCl (pH 8.4), 20

mmol/l MgCl2, and 500 mmol/l KCl,in the presence of 1U amplification grade

eoxyribonuclease 1 (DNase1) (Invitrogen) to eliminate any remaining genomic

DNA. DNase1 was inactivated by heating samples to 65C in the presence of

2.5mM EDTA to protect the RNA from Mg2+

-dependent hydrolysis. Half of the

resulting sample was reverse transcribed at 42C using 200 ng of random

hexamer primers, 400 units of Superscript III reverse transcriptase (Invitrogen)

and 40 units of RNaseOut (Invitrogen) supplemented with 2 mM dNTP, 0.1 mM

DTT, and 1X first strand buffer (Invitrogen) in a final reaction volume of 50 l.

The remaining half of the RNA sample was prepared as a negative control

without the addition of reverse transcriptase, following the same protocol.

Finally, enzymatic reactions were stopped by sample incubation at 70C for 15

min. The cDNAs were either used directly for PCR amplifications or stored at -

20C.

2.4.4 Polymerase Chain Reaction (PCR)

Primers: The primer sequences were designed to span exon-intron

borders (where possible) and were between 20-30 bp. The annealing

temperatures were 55-65oC (Table 2.4). 1-3 l of each cDNA sample were

included in 25l GoTaq MM (Promega) containing 200nM of specific primers,

1.5mM MgCl2, 200 M dNTPs, 1.25 units Taq polymerase and loading dye

(Promega). Samples were amplified using a thermal cycler (either Primus 25

42

Advanced® or Primus 96 Plus®; PEQLAB Biotechnologie GmbH, Erlangen,

Germany) with PCR profile: 94C for 2 mins, followed by 35 cycles of 94

C for

40sec, appropriate annealing temperature for 40sec, and 72C for 60sec/kb of

product size, with a final extension step of 72C for 10min. In each case, the

annealing temperature adopted was usually 1-3C below the lowest melting

temperature of the primer pair. The PCR products (10 l) were resolved on

agarose gels and the rest stored at -20

C. The size of PCR amplicons was

compared against size markers and their specificity confirmed either by

sequencing or following restriction enzyme digestion.

Control primers, like glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

were used to ensure equivalent amounts of first strands being used.

Quantitative PCR (qPCR), and semi-quantitative ‘manual’ qPCR

(mqPCR): Quantitative (Taq man qPCR) and semi-quantitative (mqPCR)

analyses were performed using an ABI Prism 7700 sequence detection system

(PerkinElmer Life Sciences) with Taqman probes. Relative expression was

calculated as a ratio of P2X7 to GAPDH. Primers (Mm01199501_m1 to mouse

P2X7 exon 3-4 and Mm00440582_m1, exons 5-6; mouse GAPDH endogenous

control; Applied Biosystems) were used according to manufacturer‟s

instructions. Semi-quantitative experiments to compare relative levels of

P2X7(a) and P2X7(k) transcripts were performed by removing 5l aliquots of

PCR reactions at cycle # 14,17,20,23 and 26 for GAPDH, and 26, 29, 32, 35 and

40 for P2X7. PCR products were resolved by agarose gel electrophoresis and

imaged following 2s UV exposure using CHEMI GENIUS2BIO imaging system

(Syngene). Bands representing PCR products were quantified using the

integrated pixel density measurement function of ImageJ software. Integrated

densities were plotted against cycle number to generate PCR profiles using non-

linear curve fitting (Origin 7.0). Ct values for GAPDH and P2X7 were derived

43

from a standardized base-line and relative expression calculated as above. All

experiments were repeated at least 3 times and performed in triplicate.

2.4.5 Agarose gel electrophoresis

PCR products could be loaded directly into wells formed in 2% agarose

gels containing 0.001% (v/v) ethidium bromide (EtBr) along DNA ladders (100

bp or 1kb, Invitrogen Ltd.) used for size reference. These samples were resolved

by electrophoresis (5 V/cm) for ~30-50 min or until required separation of bands

occurred. For testing Total RNA quality, 0.2-0.5l was loaded into 1% (w/v)

agarose gels containing 0.001% (v/v) EtBr with 1ul 10% (v/v) glycerol

replacing the loading dye. Images of fully resolved agarose gels were captured

using the CHEMI Genius2 Bio imaging System (Syngene Ltd).

2.4.6 Restriction digests

Specific restriction enzymes for digesting the DNA fragment of interest

were identified using the freeware, Sequence Manipulation Suite (SMS, Version

2.0), available from the website of Bioinformatics Organisation Inc

(Massachusetts, USA). The chosen restriction enzymes (preferably single or

double cutters) were added at concentrations of 2-5 units into an appropriate 1x

buffer or directly into the PCR product (if a particular enzyme was active in the

1x PCR buffer). Otherwise, the PCR products were first purified using the

MinElute PCR purification kit according to manufacturer‟s instructions.

Alternatively, the specific PCR product band in agarose gel slices was extracted

with the MinElute gel extraction kit (50) Qiagen. A 0.5 μl from these purified

samples was used for restriction enzyme digestion at 37C for 2 hr.

44

Subsequently, the enzymes were inactivated at 70C for 10 min before resolving

the digested samples on 2% (w/v) agarose gels or using in cloning or other

downstream applications.

2.4.7 TA cloning of PCR products

Fresh PCR products (3 μl) were ligated into 50 ng of pGEM®-T Easy

Vector (Promega) in a reaction buffer containing 1X rapid ligation buffer, and 3

units of T4 DNA Ligase (Promega). The ligation process was performed either

at room temperature for 2 hr or at 4C overnight. PCR products kept for more

than 3 days at -20C need dA-overhangs refreshed prior to ligation. For this, 3 μl

of PCR product were incubated at 70C for 30 min in a 10 μl reaction buffer

containing 1X PCR buffer, 5 units of Taq DNA Polymerase (Qiagen), and 0.2

mM dATP (Invitrogen).

Transformation of Ligated Vector into JM109 Competent Cells: Ligation

reaction mixtures (10 μl) were introduced into 200 μl of JM109 competent cells

(Promega), on ice. JM109 competent cell/ligation mix mixtures were heat-

shocked at 42C for 45-50 sec in 14ml polypropylene round bottom tubes

(Falcon #352059) and returned immediately to ice for a further 2 min.

Transformed JM109 competent cells were mixed with 500 μl of SOC medium

(Promega) pre-equilibrated to room temperature. These mixtures were left in a

shaking incubator (37C, 150rpm) for 1.5 hr. 100 μl of transformed JM109 were

spread (under aseptic conditions) onto LB agar plates supplemented with 50

μg/ml ampicilin, 0.5 mM IPTG, and 80 μg/ml X-Gal. JM109 competent cells

from the remaining SOC mix were pelleted at 800g for 10 min. The resulting

transformed cell pellets were re-suspended in 200μl of fresh SOC medium, of

which 100μl was spread on LB agar plates, thereby creating a concentrated

45

version of the above. Inoculated LB agar plates were incubated upside-down at

37C overnight.

Blue/White Recombinants Colony Screening: a few blue and white colonies

on LB agar could be expected after an overnight incubation at 37C. Those

positive ones (white colonies; preferably surrounded by some blue) were picked

up with the aid of sterile toothpicks, which were then briefly dipped in prepared

PCR reaction mixtures (containing gene-specific primer pairs), and then

dropped into 3 ml LB broth with added 50 μg/ml ampicillin. The inoculated LB

broths were shaken (37C, 220rpm) for 6-8 hr at 37

oC and subsequently

centrifuged at 2,300g for 5 min. Plasmid DNA were later extracted from the

pelleted bacterial cells. In the meantime, the PCR amplification results would

have indicated those colonies, which were likely to contain the plasmid clones

of interest.

Extraction of Plasmid DNA: Plasmid DNA was extracted using the QIAprep®

Miniprep Kit (Qiagen Ltd.), according to the manufacturer‟s instructions.

Briefly, pelleted bacterial cells were re-suspended in 250μl of buffer P1 (with

the RNase A provided). Cells were lysed following the addition of 250μl of

buffer P2 with gentle mixing. Unwanted cell debris were immediately

precipitated by adding 350μl of buffer N3 and pelleted at 15,600g for 10 min.

The resulting supernatants were transferred onto the QIAprep Spin Column

(Qiagen) and passed twice through the silica-gel membrane within by

centrifuging at 15,600g for 1 min. Plasmid DNA trapped on the membrane was

washed with 750μl of buffer PE drawn through the membrane bycentrifugation

at 15,600g for 1 min. A further 2 min centrifugation was used to remove any

residual ethanol present in buffer PE. Purified plasmid DNA was eluted from

the silica-gel membrane into fresh micro-centrifuge tube by soaking the

membrane with 30μl of nuclease free water or TE buffer followed by

centrifugation at 15,600g for 1 min. This step was repeated again for increased

46

DNA yield. A small aliquot of extracted plasmid DNA was digested with

appropriate restriction enzymes to confirm that expected clones were obtained.

5μl of the plasmid DNA was sent for commercial DNA sequencing (Lark).

2.4.8 Protein extraction

Adherent cell cultures: Proteins were extracted on ice by scraping cells

(Cell Scraper, Greiner) in a minimal volume of extraction buffer composed of:

1x LysisM extraction buffer (Cat. #04719956001, Roche), 1x protease inhibitor

cocktail tablet (Roche) per 10ml LysisM, 2x phosphatase inhibitor cocktail

tablets (Roche) / 10ml LysisM, 2mM sodium orthovanadate. Samples were

homogenised by passing through a 23g needle 20 times, then stored at -20C.

Tissues: Proteins were extracted from frozen tissues by crushing a known

weight of sample into a fine powder in liquid nitrogen using a mortar and pestle

(pre-chilled on dry ice). The tissue powders were transferred into a 15 ml

centrifuge tube (Fisher), any excess liquid nitrogen allowed to evaporate on ice.

A minimal volume of extraction buffer was added (composition as above) and

samples were further homogenised by passing through a 23g needle 20 times,

then stored at -20C.

2.4.9 Protein concentration assay

Protein concentrations of extracted samples were determined using the

bicinchonic acid (BCA) method (Sigma-Aldrich Ltd.). 1-5ul of protein samples

were used for each triplicate measurement. Protein standards comprised of

bovine serum albumin (BSA) (Sigma-Aldrich Ltd.) in the range 0-10ug.

Standards and samples were added to a 96 well plate (Greiner), individually

47

mixed with 200ul of BCA working reagent (prepared by mixing 50 parts of

BCA solution and 1 part of 4% (w/v) CuSO4.H2O). The resultant mixtures were

incubated at 55C for 15 min to allow blue colouration to develop. Absorbance

was then measured at 550 nm using a plate reader (Antos 2001). Protein

concentrations (ug/ul) were obtained by deriving the equation of line of best fit

from average standard values according to y=mx+c.

2.4.10 SDS polyacrylamide gel electrophoresis (SDS-PAGE)

Gel casting: Mini-PROTEAN 3 casting frame, casting stand, and gel

cassette (Bio-Rad Laboratories Ltd.) were assembled according to

manufacturer‟s instructions. Resolving gels were of composition 6-12% (v/v)

acrylamide/bisacrylamide, 0.375 M Tris-HCl (pH 8.8), 0.1% (v/v) sodium

dodecyl sulphate (SDS), 0.05% (v/v) ammonium persulfate (APS), and 0.01%

(v/v) tetramethylethylenediamine (TEMED). Gels were poured, over-laid with

water and set for ~30 mins, before the water was removed and stacking gels of

composition 4% (v/v) acrylamide/bisacrylamide, 0.125 M Tris-HCl (pH 6.8),

0.1% (v/v) SDS, 0.05% (v/v) ammonium persulfate (APS), and 0.01% TEMED

(v/v) were poured on top of the resolving gel. Combs (Bio-Rad Laboratories

Ltd.) were inserted and gels set for ~5 mins. Gels were usually used

immediately but sometimes stored for up to 5 days at 4C wrapped in wet paper

towels and Clingfilm.

Sample preparation for electrophoresis: Protein samples were mixed with

Laemmli sample buffer (62.5 mM Tris-HCl, 2% (v/v) SDS, 25% (v/v) glycerol,

and 0.01% (w/v) bromophenol blue) at 1:1 protein/buffer ratio. Occasionally, a

2:1 protein/buffer ratio was adopted to further maximise the amount of proteins

being loaded in a well. Subsequently, these mixtures were supplemented with

48

2.5% (v/v) -mercaptoethanol and boiled for 5 min at 95C. Denatured protein

samples were briefly chilled on ice before gel loading.

Electrophoretic separation of proteins: Mini-PROTEAN 3 Gel Cassette and

electrophoresis module (Bio-Rad Laboratories Ltd.) was assembled according to

the manufacturer‟s instructions. 20-40l of each denatured protein sample were

loaded and resolved at150-200V, 100mA in 6-12% (w/v) SDS-polyacrylamide

gels for ~120 min in electrophoresis buffer (25 mM Tris, 192 mM glycine, and

0.1% (w/v) SDS, pH8.3). 5-10l of prestained molecular weight markers (Lonza

Ltd.) was resolved alongside the protein samples serving as a reference for

approximate protein size determination. Resolved protein gels were either

transferred to polyvinylidene fluoride (PVDF) membranes (Amersham

Biosciences Ltd) or stained with coomassie brilliant blue G (CBB) solution

(Sigma-Aldrich Ltd.) to assess protein sample quality / amount / quality of gel

running.

Coomassie Brilliant Blue G staining of gels: Resolved protein gels were

submerged in CBB solution [0.1% (w/v) CBBG, 25% (v/v) methanol, and 5%

(v/v) glacial acetic acid] for 30 min. The stained protein gels were washed three

times in de-stain solution (7.5% (v/v) methanol and 10% (v/v) glacial acetic

acid) for 15 min. With the final change of fresh destain solution, the protein gels

were left to destain overnight covered with a piece of paper towel to soak up the

released dye.

2.4.11 Electrophoretic protein transfer

Trans blot assembly: Mini trans-blot cell and gel holder cassettes (Bio-

Rad Laboratories Ltd.) were assembled according to the manufacturer‟s

instructions. PAGE protein gels were placed onto PVDF (Hybond P, Amersham

Bioscience Ltd)membrane pieces cut to the same size as the gel. PVDF

49

membrane, was pre-treated in absolute methanol for 5 sec, then washed in 1x

Transfer buffer (25 mM Tris, 192 mM glycine, and 20% (v/v) methanol; pH 8.3)

for 5 min before use. Both gel and membrane were then sandwiched between

buffer-soaked filter papers, fiber pads, and gel holder cassettes. Proteins were

transferred onto the blotting membrane through electrophoretic transfer

(conditions: 100 V; 350 mA) for 2 hrs at 4C using pre-chilled transfer buffer.

Following electrophoretic transfer, blots were trimmed to size and submerged in

blocking buffer [5% (w/v) non-fat milk powder in 1x PBS and 0.01% (v/v)

Tween-20 (Sigma-Aldrich Ltd.)] for 1 hour prior to probing with antibodies.

2.4.12 Antibody immunoblotting

Blots were probed with relevant primary antibody diluted at the optimal

concentration in the same blocking buffer overnight at 4C or at RT for 2 hrs.

Blots were then washed three times with 1x PBST for 10 min, and then

incubated with the appropriate horseradish peroxidase-conjugated secondary

antibody (Amersham Biosciences Ltd.) for 45 min. Following a further three, 10

min washes in 1x PBST, specific protein bands were visualized using luminol-

based substrates (Uptilight/Uptilight US, Cheshire Sciences). Images were

obtained using a G:BOX Chemi XT-16 system (Syngene). Used blots could be

stipped by incubating in stripping buffer [50 mM Tris-HCl (pH 6.8), 2% (v/v)

SDS, and 2 mM β-mercaptoethanol] for 10 min at 50C, followed by 3 washes

with 1x PBST for 5 min. Blots could then be re-probed with another antibody.

The stripped blots could otherwise be sandwiched between filter paper sheets to

store dry for later use.

50

2.4.13 Antibodies:

Primary antibodies, their sources and dilutions used are summarised in

Table 2.3.

Actin: Anti-Actin (Sigma-Aldrich Ltd. Cat #A2066) is an affinity purified

rabbit polyclonal antibody that has been designed to recognise the carboxyl

terminal portion of almost all (α, β, and γ) actin isoforms. It recognises a 42

KDa actin band in different animal tissue extracts using WB (diluted 1:1000),

this antibody was used for assessing the qualities and quantities of protein

loadings.

Desmin: Anti-desmin clone D33 (Dako Ltd. Cat. #MO760) mouse monoclonal

antibody labels the intermediate fiolament protein desmin in smooth and skeletal

muscle cells. Used at 1:200 for immunocytochemistry.

Dystrophin: Anti-dystrophin 2166 (Gift from Prof Derek J Blake, Cardiff) is

affinity purified goat polyclonal antibody which recognises a specific N-

terminal sequence of the full length muscle-type dystrophin isoform (Dp427).

The antibody was used at 1:400 dilution for immunohistochemistry. Anti-

dystrophin 7A10 (Developmental Systems Hybridoma Bank, University of

Iowa) is mouse monoclonal antibody recognising a specific C-terminal sequence

of dytrophin present in all known dystrophin isoforms. The antibody was used

at 1:100 for WB.

ERK1/2 and phospho-ERK1/2: Anti-ERK1/2 (Cell Signalling Cat. #9102) is

a rabbit polyclonal and anti phospho-ERK1/2 (Cell Signalling Cat. #9106) is a

mouse monoclonal antibody. These were used in WB (diluted 1:2000 and

1:1000 respectively) and recognised ERK bands of 44 and 42KDa.

Phosporylated forms are undistinguishable by size and so antibodies were used

separately to probe the same protein extracts.

51

F4/80: Anti-F4/80 pan-macrophage marker (Abcam Cat. #74383) is rabbit

polyclonal antibody. In WB (diluted 1:500) this antibody recognises a specific

band of ~130KDa plus an additional band of 30KDa of an unknown origin.

P2X7: Anti-P2X7 (Cell Signalling Ltd. Cat # 177003) is affinity purified rabbit

polyclonal antibody raised against the C-terminal portion of the wild type

variant. In WB (diluted 1:500) it recognises a specific 80KDa band and a

possible un-glycosylated lower molecular weight variant of ~70KDa.

Biotin-conjugated secondary antibodies: Both biotinylated rabbit anti-goat

IgG (Cat #BA-5000) and goat anti-rabbit IgG (Cat #BA-1000) were used at

1:200 dilutions. For IHC applications these secondary antibodies were used in

conjunction with either VectaStain Elite ABC kit (peroxidase-based; Cat #PK-

6100) or VectaStain ABC-AP kit (alkaline phosphatase-based; Cat #AK-5000),

according to manufacturer‟s instructions. Peroxidase (DAB or Vector VIP) and

alkaline phosphatase (Vector Blue) substrates were applied to visualise the

stains. For IF applications, Fluorescein avidin D (Ex/Em: 495/515 nm; Cat #A-

2001) or Texas red avidin D (Ex/Em: 595/615 nm; Cat #A-2006) were used with

those biotin-conjugated secondary antibodies (All from Vector Laboratories

Ltd.).

Fluorophore-conjugated secondary antibodies: All fluorophore-conjugated

secondary antibodies were obtained from Molecular Probes Inc., Eugene, OR.

These included Alexa Fluor 488 goat anti-rabbit IgG (Ex/Em: 495/519 nm; Cat

#A-11008), Alexa Fluor 488 chicken anti-mouse IgG (Cat #A-21441), and

Alexa Fluor 546 donkey anti-goat IgG (Ex/Em: 556/573 nm; Cat #A-11056).

These were used in IF at 1:200 dilutions for all applications including on

cultured cells or tissues sections.

Horseradish peroxidase-conjugated secondary antibodies: Affinity purified

horseradish peroxidase-labelled antibodies, goat anti-rabbit IgG (Cat #A0545)

52

and rabbit anti-goat IgG (Cat #A5420), were bought from Sigma-Aldrich Ltd.,

Dorset, UK. These secondary antibodies were used for WB at 1:10,000

dilutions.

2.4.14 Tissue processing techniques

Dissection of mouse tissues: Mice (4 months old) were killed by

schedule 1 method and their tissues carefully dissected out. Excised tissues

were either flash-frozen for proteins extraction by dropping into liquid nitrogen

in sealed Eppendorfs for storage at -70C or processed for cryo-sectioning.

Processing tissues for cryo-sectioning: Freshly isolated organs were orientated

and embedded in a dab of cryo-embed (R.A. Lamb Ltd.) on a 20mm diameter

cork disc (R.A. Lamb Ltd.) prior to being inverted and submerged in a beaker of

isopentane chilled in liquid nitrogen. Mouse brains were embedded in cryo-glue

and left to slow-freeze on a flat metal surface placed on dry ice. Frozen tissues

were then stored at -70C until needed for cryo-sectioning.

Samples on cork disks were transferred into a cryostat chamber (Bright OTF

Cryostat; Raymond A Lamb Ltd. Cat No. E7.15/OTF). These were left to

equilibrate at -20C for ~1 hr before cryo-sectioning. Tissue samples were

mounted and 10 m thick sections cut. Sections were collected onto poly-L-

lysine-coated slides, air-dried and stored at –70C.

Fixation of tissue samples: 4% (w/v) paraformaldehyde in 1x PBST was the

preferred fixative used for post-fixation of tissue sections. Slides with tissue

sections were incubated with the fixative for 15 min on ice.

53

2.4.15 Immunolocalization

In order to aid antibody specificity high salt concentration (2.5% (w/v)

sodium chloride) was employed during dystrophin antibody incubations, which

had the effect of reducing non-specific background staining from the secondary

antibody. Negative controls, performed using secondary antibody without

primary antibody were utilised to assess the presence of any non-specific

background.

Immunocytochemistry: Cells were fixed in either chilled absolute methanol or

4% (w/v) paraformaldehyde (PFA) in 1x PBS for 15 min at 4C. The latter

required the cells to be permeabilised, hence, 0.5% (v/v) Triton X-100 was

added to the appropriate 10% (v/v) normal serum in 1x PBS, and the mixture

was subsequently applied to the cells for 30 min. Further blocking with avidin

and biotin blocking solutions (Vector Laboratories Ltd.), 15 min each, was

necessary for colorimetric staining protocols.

Colorimetric immunocytochemistry: The cells were washed with 1x PBS for 5

min after every incubation step, then incubated overnight at 4C or at RT for 2

hrs with the specific primary antibody (diluted using 10% (v/v) normal serum in

1x PBS). Cells were washed for 5 min each with 1x PBS prior to incubation

with the respective biotinylated secondary antibody (diluted at 1:200 with 2%

(v/v) normal serum in 1x PBS) for 45 min. The cells were visualised following

incubation with Vectastain ABC-AP kit (Vector Laboratories Ltd.) for 30 min,

followed by its respective alkaline phosphatase substrate (Vector Blue

substrate kit; Vector Laboratories) until the desired colouration was achieved. A

brief 5 min counterstaining in methyl green (Vector) was also used. Finally, the

cells were coverslipped using Faramount® aqueous mounting medium

(DakoCytomation).

Immunofluorescence: The cells were washed with 1x PBS for 5 min after every

incubation step, then incubated overnight at 4C or at RT for 2 hrs with the

54

specific primary antibody (diluted using 10% (v/v) normal serum in 1x PBS).

Cells were washed three times for 5 min each with 1x PBS prior to incubation

with the respective Alexa Fluor (Invitrogen) 488-conjugated chicken anti-rabbit

IgG (1:200), 488-conjugated goat anti-rabbit IgG (1:200), or Alexa 546-

conjugated donkey anti-goat IgG (1:200), plus Hoechst (1:1000) as a nuclear

counterstain. Sections were mounted in non-fluorescing Vectashield mounting

medium (Vector Laboratories Ltd). Labelling was imaged using an Axioplan 2

imaging MOT microscope (Carl Zeiss, Jena, Germany) and specific sections

were further analysed using the confocal laser scanning module, LSM 510

META (Carl Zeiss).

Immunohistochemistry: Tissue sections obtained by cryosectioning were post-

fixed with cold 4% (w/v) PFA in 1x PBS for 15 min. Sections were treated with

0.3% (v/v) hydrogen peroxide in methanol for 30 minutes to eliminate any

endogenous peroxidase activity, then blocked in appropriate 10% (v/v) normal

serum in 1X PBS for 30 min and subsequently with avidin/biotin blocking kit

(Vector Laboratories Ltd.) for 15 min per step if colorimetric staining protocols

were used. Brief 5 min washes in 1x PBS were incorporated between each step.

Next, tissue sections were incubated overnight at 4C or at RT for 2 hrs with the

specific primary antibody at optimised dilution prepared using the same

blocking solution as above. Appropriate biotinylated secondary antibody

(Vector) diluted at 1:200 with 2% (v/v) normal serum in 1x PBST, and

subsequently Vectastain ABC reagent (Vectastain Elite ABC Kit; Vector

Laboratories Ltd.) were applied (30 min each). Sections were rinsed three times

for 5 min each in PBS after each incubation step. Staining was visualised by

incubating the sections in a peroxidase substrate (Vector VIP or DAB substrate

kits, Vector Laboratories Ltd.) until the desired colour intensity was reached.

Sections were then counterstained with methyl green (Vector), dehydrated in

ascending ethanol series (50-100%) into xylene, and mounted in dipthyline

55

xylene (DPX). For immunofluoescence staining, biotinylated antibodies were

exchanged for Alexa Flour (Invitrogen Ltd) 488-conjugated chicken anti-rabbit

IgG (1:200), 488-conjugated goat anti-rabbit IgG (1:200), or Alexa 546-

conjugated donkey anti-goat IgG (1:200), plus Hoechst (1:1000, Sigma) nuclear

counterstain. Sections were mounted in non-fluorescing Vectashield mounting

medium (Vector). Labelling was imaged using an Axioplan 2 imaging MOT

microscope (Zeiss) and specific sections were further analysed using the

confocal laser scanning module, LSM 510 META (Zeiss).

2.4.16 Histological staining

Haematoxylin and eosin staining: Tissue sections were fixed on ice in

cold 4% PFA for 15 min, and then incubated in haematoxylin solution (Sigma)

for 1 min. Acid alcohol (1% (v/v) HCl in 70% ethanol) was used for the

differentiation of tissue sections before finally counterstaining them with the

eosin solution (Sigma) for a further 1 min. The sections were dehydrated through

ascending ethanol series (50-100%), cleared in xylene and mounted in DPX

under coverslips.

Immunolocalisation of the revertant fibres: Two 10 m thick muscle cryostat

sections were taken from approximately equal isobaric locations (one third and

two thirds longitudinal distance from ankle to knee) in each muscle examined.

Sections were dried onto poly-L-lysine coated glass microscope slides (Menzel

Gläser), fixed with ice-cold 4% paraformaldehyde in PBS for 10 min and

blocked in 10% v/v normal goat serum (NGS) in PBST (0.01% v/v Tween-20)

for 30 min at RT. After blocking, the sections were incubated with the

dystrophin antibody in PBST containing 2% v/v NGS, washed extensively in

PBS and visualised either via immunofluorescent detection with Alexa Fluor

488-labelled secondary antibody and confocal analysis performed using an LSM

56

710 microscope (Zeiss) or via the histochemical detection using Vectastain

ABC-AP kit (Vector). In the latter method, secondary antibody incubation for 30

min was followed by its respective alkaline phosphatase substrate (Vector

Blue substrate kit; Vector), until the desired colouration was achieved. Centrally

nucleated fibres were visualised by DAPI and methyl green staining for confocal

and histochemical staining, respectively. For negative controls the primary

antibody was omitted in the staining procedure. Labelled muscle sections were

imaged using LSM 710 (Zeiss) with HD colour camera, then assembled as

macro-images (Corel PHOTO-PAINT X3), which were used for binary label

based counting (Origin 7.0, cell-counter plug-in). Data was presented as number

of centrally nucleated / revertant fibers as a percentage of total fiber number per

stained cross sectional area.

2.4.17 ATPe hydrolysis assay - Measurement of inorganic phosphate (Pi)

release

The following protocols are based on the colorimetric shift of phospho-

molybdate complexes upon their reduction in acidic solutions. Two such

methods were tested here, using different reducing agents, which included

Malachite green hydrocloride and L-ascorbic acid. The latter was rejected in

this instance; although this assay proved more sensitive over a greater linear

range than the Malachite green-based method, a greater dependence upon the

correct emission filter was encountered when using the L-ascorbic acid method.

In this instance, the only available suitable emission filter was 600nm. With

both methodologies trialled here requiring a 655nm emission filter, the

flexibility of this parameter proved the deciding variable in assay selection,

which led to the adoption of the Malachite green-based Pi detection assay.

Should the correct 655nm emission filter be available in microtiter plate format,

57

the L-ascorbic acid method would be considered a more accurate and adaptable

assay for small volume, low concentration Pi detection.

Malachite Green-based assay: The extracellular ATP-hydrolysing

potentials of C2C12, IMO and SC5 cell cultures were assessed using a modified

version of the Malachite green method (Lanzetta et al., 1979). Myoblasts were

cultured in 12-well plates as described above; cells were seeded at 50%

confluency and experiments conducted on the second day of culture, when cells

were 70-80% confluent, or where myotubes were used, after a further 4 days in

differentiation medium. Cells were washed twice with 500µl of phosphate-free

buffer (140mM NaCl, 20mM Hepes, 4mM MgCl2, 2mM CaCl2, pH7.8) then

incubated in 500µl of the same buffer containing 0–4mM ATP. Aliquots of the

incubation medium (20µl for myotube, 50µl for myoblast cultures) were

removed at time T=0 and T=30 mins following treatment with ATP for

inorganic phosphate (Pi) detection. Samples were transferred to 150ul solution

A (Two parts 0.045% w/v Malachite green and 0.002% v/v Triton-N101 in

ddH2O to 1 part 4.2% w/v ammonium molybdate in 4M HCl), which was mixed

by rotation for 1 hour and filtered to remove debris prior to use (11µM,

Whatman Grade 1). After 1 min, 50µl 2% sodium citrate was added to halt

further colour development. Solutions were incubated for a further 15mins at

RT, allowing for colour stabilisation prior to reading of absorbance values at

600nm using a POLARstar Optima microplate reader (BMG Labtech). Triton-

N101 replaced Sterox (Lanzetta et al., 1979) as the Pi solubilising agent here due

to the lack of commercially available Sterox. Triton-N101 proved to contain the

lowest level of Pi of the laboratory grade detergents tested, e.g. NP-40, SDS,

CHAPS, Triton-X-100, most of which contained sufficiently high levels of Pi to

quench any ATP derived Pi. Phosphate standards (0-500µM KH2PO4 in ddH2O)

were prepared fresh and assayed alongside treated samples. Extracellular ATP

hydrolysis was calculated by subtracting Pi at t = 0 from Pi at t = 30 min and

58

values were normalised against the protein content of each preparation (BCA

protein assay, Sigma – see 2.4.9). Extracellular ATP hydrolysing potentials

were compared by determining KM and Vmax values were calculated from the

fitting of saturation curves to Pi release data in response to 0-4mM ATP using a

one-site curve fitting function (Microcal Origin7.03). Differences between KM

or Vmax values were tested for significance by two-way ANOVA and Tukey‟s

post hoc test.

L-ascorbic acid-based assay: The culturing, preparation and treatment

of cultured cells did not differ between Malachite green- and L-ascorbic acid-

based protocols – these protocols differed only in the reducing agent used for Pi

detection. See the above Malachite green-based assay methodology for detailed

protocol relating to the culturing of cells for this assay. As with the above

protocol, 50ul aliquots of incubation media were removed at time T=0 and

T=30mins following treatment with ATP for inorganic phosphate (Pi) detection.

Aliquots were transferred to 150ul solution A (Two parts 12%w/v L-ascorbic

acid in 1M HCl to one part 2% w/v ammonium molybdate tetrahydrate in ddH2O

– this solution was prepared fresh and not kept longer than 1 hour at RT).

Following 5min incubation at RT, 100ul solution B (2% sodium tribasic

dehydrate and 2% acetic acid in ddH2O) was added to prevent further colour

development. Solutions were incubated for 15mins at RT to allow for colour

stabilisation prior to reading of absorbance values at 600nm using a POLARstar

Optima microplate reader (BMG Labtech). Kinetic parameters were determined

as per Malachite Green-based assay protocol above.

59

2.4.18 ERK1/2 phosphorylation assay

Cells were seeded at 50% confluency in 6 well collagen (Sigma) coated

plates (Greiner) in 2ml normal growth medium and left to proliferate overnight.

24 hours later, cells were washed three times in DPBS and growth medium was

replaced with low serum medium (0.5% v/v serum). Cells were incubated in

this medium for a further 12 hours prior to treatment. Agonists and antagonists

were applied for the stated time periods at the following concentrations: ATP,

500uM, BzATP, 300uM, Coomassie Brilliant Blue G (CBBG), 1uM, ivermectin

(IVM), 0.25uM. Where used, antagonists were applied for a 10 min pre-

incubation prior to agonist addition. Following treatment for the indicated time

periods, cells were lysed as described above and extracted proteins used for

immuno-blot analysis of ERK1/2 phosphorylation status by probing with

specific anti-p44/42 and anti-phosphop44/42 antibodies (see below).

2.4.19 PNGase F deglycosylation

Proteins were deglycosylated using PNGaseF (New England Biolabs)

according to the manufacturers‟ instructions. Briefly, 100g of total protein

lysate was denatured in denaturing buffer (5% v/v SDS, 0.4M DTT) for 10 mins

at 100°C, then samples were deglycosylated in reaction buffer containing 2500

U PNGaseF, 0.5M sodium phosphate, 10% NP-40, for 1 hour at 37°C. 15g of

digested samples were used for immune-blotting with anti-P2X7 antibody as

previously described. In some cases the heat step was omitted.

60

2.5 Proteomics

2.5.1 Phosphoprotein purification

5 mg of total protein extract was required for each sample to be enriched

for phosphoproteins. Cells were cultured as described in 500cm2 plates and

proteins extracted by scraping in 5ml of phospho-protein lysis buffer (25mM 2-

morpholinoethanesulfonic acid (MES), 1mM NaCl, 0.25% w/v CHAPS, 1x

protease inhibitor tablet, 1x phosphatase inhibitor tablet, 250U benzonase

nuclease, pH, 6.0). Samples were then homogenised by 20 passes through a 23g

needle. Phosphoprotein purifications were carried out using PhosphoProtein

Purification Kit (6) (Qiagen) according to manufacturer‟s instructions. Briefly,

extracted protein was left on ice for 30 min, and then centrifuged at 10,000g at

4C for 30 min, during which time phosphoprotein purification columns were

prepared to receive samples by allowing the storage buffer to flow out. Sample

supernatants were harvested and the protein concentration determined by BCA

method, as described previously. A volume of lysate containing 2.5mg of total

protein was diluted to 0.1mg/ml with phosphoprotein lysis buffer containing

0.25% w/v CHAPS. Final volumes were between 30-40ml per sample. Lysates

were applied to binding columns at RT. Columns with bound samples were

washed twice in 3ml phosphoprotein lysis buffer containing 0.25% w/v CHAPS

and samples eluted into Amicon ultra centrifugal filters (Ultracel-3K, Fisher) in

4ml phosphoprotein elution buffer (50mM potassium phosphate, 50mM NaCl,

pH 7.5). Samples were centrifugally filtered at 3,100g until volumes had been

reduced to 200l, and then stored at -80C.

61

2.5.2 Mass spectrometry

Mass spectrometry was carried out using phospho-protein enriched

samples in collaboration with Dr. Philippe Chan (University of Rouen, France).

Samples were de-salted using maxi dialysis tubes (3ml, 3.5KDa, Generon)

floating in cold ddH2O for 2 hours, then again in fresh solution overnight.

Sample pH was reduced to ~pH 6.0 by adding triethylammonium (Sigma) until

Litumus test was satisfactory. Samples were then denatured in 5mM tris(2-

carboxyethyl)phosphine (TCEP) in triethylammonium at 60C for 1 hour, then

cystein residues were blocked in 55mM iodoacetamide at RT for 30 mins.

Trypsin (V5111, Promega) digestion of samples was carried out using 1g

trypsin per 1g protein at 30C for 15 mins. Samples were labelled with iTraq

labels from 114-117 (Applied Biosystems) dissolved in EtOH for 1 hour at RT.

Labelled samples were subjected to isoelectric focussing in a 24 well 12.5% w/v

poly-acrylamide gel strip (Agilent Technologies) placed in an OFFGEL

fractionation system (Agilent 3100 Fractionator, Agilent Technologies) for ~48

hrs. Separated samples were analysed in an „ESI-LC-MS‟ (Agilent 6200 TOF,

Agilent Technologies) and phosphopeptide fragments identified by interrogating

the „Mascot‟ database (Matrix Science) using „Spectromill‟ (Agilent

Technologies). Fold changes in phosphoprotein levels between treated, normal

and dystrophic myoblasts were calculated using the „iQuantitator‟ package

(2009) in the „R‟ statistical environment (R Development Core Team, 2010).

62

2.6 Statistical analysis

Statistical significance of data was assessed using one- or two-way

analysis of variance (ANOVA) with post-hoc Tukey‟s test (Origin 7.0) or

Bonferonni test (Prism 4) respectively. P<0.05 was considered statistically

significant, data are reported as means ± SE, where n=3-5 for pharmacological

assays, plus PCR and Western data, and n=3-9 for histological analyses of

muscles. „Degrees of freedom‟ (df) is used throughout in place of „n‟, where

df=n-1.

63

Table 2.2 Compositions of buffers, stains and solutions

Buffer/Stain/Solution Composition

Acid Alcohol 1% (v/v) HCl in 70% ethanol

Blocking Buffer 5% Low fat dried milk powded

Coomassie Brilliant

Blue G Stain

0.1% (w/v) CBBG, 25% (v/v) methanol, and

5% (v/v) glacial acetic acid

De-stain Solution 7.5% (v/v) methanol and 10% (v/v) glacial

acetic acid

Electrophoresis

Buffer

25 mM Tris, 192 mM glycine, and 0.1% (w/v)

SDS

KC Solution, 1x 0.041g K3Fe(CN)6, 0.0525g K4Fe(CN)63H2O,

1.25ml 1X PBS, pH 5.0

Laemmli Sample

Buffer

62.5 mM Tris-HCl, 2% (v/v) SDS, 25% (v/v)

glycerol, and 0.01% (w/v) bromophenol blue

Lysis Buffer 10ml 1X LysisM (Roche), 1X protease inhibitor

tablet (Roche), 2X PhosSTOP inhibitor tablets

(Roche), 2mM Na3VO4

PBS, 1X 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4,

and 2 mM KH2PO4

PBST, 1X 1X PBS and 0.01% (v/v) Tween 20

PBS/MgCl2 1mM MgCl2 in PBS, pH 5.0

Stripping Buffer 50 mM Tris-HCl, 2% (v/v) SDS, and 2 mM β-

mercaptoethanol

TBS, 1X 50 mM Tris.HCl, 150 mM NaCl, pH 7.4

TBST, 1X 1X TBS and 0.01% (v/v) Tween 20

TAE, 1X 40 mM Tris, 1 mM EDTA, and

0.1% (v/v) glacial acetic acid

Transfer Buffer 25 mM Tris, 192 mM glycine, and

20% (v/v) methanol (+/-0.01% SDS depending

on gel percentage).

64

Table 2.3 Primary antibodies

Antigen Antibody Species Supplier Dilutions

P2X7 177003 Rabbit Synaptic Systems WB/IHC/ICC,

1:500

Dystrophin 2166 Rabbit Gift from Dr.

Derek J Blake

IHC, 1:500, IF,

1:250

Dystrophin 7A10 Mouse Developmental

Systems

Hybridoma Bank

(DSHB)

WB, 1:100

Desmin D33 Mouse Dako ICC, 1:200

ERK1/2 9102 Rabbit Cell Signalling WB, 1:2000

pERK1/2 9106 Mouse Cell Signalling WB, 1:1000

F4/80 74383 Rabbit Abcam WB, 1:500

Actin A2066 Rabbit Sigma Aldrich WB, 1:500

65

Target Primer Sequence, Melting temperature

-SG 562-582 -SG Fv 5‟-CGAGGTCACAGCCTACAATCG-3‟, Tm, 73C

-SG 890-871 -SG Rv2 5‟-CCATCTTCAGGCAGGTGGAG-3‟, Tm, 72C

Dystrophin micro-

hinge

Hinge Fv 5‟-GCTTTGGTGGGAAGAAGTAGAGGAC-3‟, Tm, 68C

Dystrophin ex65 Ex65 Rv 5‟-CTGCAGGATATCCATGGGCTGTC-3‟, Tm, 71C

Dystrophin

mdx allele

P9427 Fv 5‟-AACTCATCAAATATGCGTGTTAGTG-3‟, Tm, 60C

Dystrophin

mdx allele

P259E Rv 5‟-AATTTACCGAAGTTGATAGACTCACTG-3‟, Tm, 60C

Dystrophin

WT allele

P260E Rv 5‟-GATTTACCGAAGTTGATAGACTCACTG-3‟, Tm, 60C

Neomycin

cassette

Neo Fv 5‟-CTTGGGTGGAGAGGCTATTC-3‟, Tm, 60C

Neomycin

cassette

Neo Rv 5‟-AGGTGAGATGACAGGAGATC-3‟, Tm, 60C

P2X7 exon 13 Pfz Fv 5‟-TGGACTTCTCCGACCTGTCT-3‟, Tm, 60C

P2X7 exon 13 Pfz Rv 5‟-TGGCATAGCACCTGTAAGCA-3‟, Tm, 60C

P2X7 exon1-2 Glaxo A 5‟-TGCCCATCTTCTGAACACC-3‟, Tm, 60C

P2X7 exon1-2 Glaxo C 5‟-CTTCCTCTTACTGTTTCCTCCC-3‟, Tm, 60C

P2X7 exon1-2 Glaxo E 5‟-GCAAGGCGATTAAGTTGGG-3‟, Tm, 60C

P2X7 exon1 mX7ex1 Fv 5‟-CACATGATCGTCTTTTCCTAC-3‟, Tm, 60C

P2X7 exon1 mX7ex1‟ Fv 5‟-GCCCGTGAGCCACTTATGC-3‟, Tm, 60C

P2X7 exon4 mX7ex4 Rv 5‟-GCACAGTGCTCTTCTGACC-3‟, Tm, 60C

P2X7 exon7 mX7ex7 Rv 5‟-GCAGGAGAGAACTTTACAGA-3‟, Tm, 60C

P2X7 exon9 mEx9 Fv 5‟-GAGAACAATGTGGAAAAGCGG-3‟, Tm, 66C

P2X7 exon11 mEx11Rev 5‟-TGACTTGCTCATCAACACATACTCCAG-3‟, Tm, 67C

P2X7 exon13 P2X7 13 WT 5‟-TCAGTAGGGATACTTGAAGCC-3‟, Tm, 60C

P2X7 exon13(c) P2X7 (13c) 5‟-TCAGGTGCGCATACATACATG-3‟, Tm, 60C

P2X7 exon13(b) P2X7 (13b) 5‟- TCTGTGAGAAACAAGTATCTAGGTTGG-3‟, Tm, 60C

Primers used as controls

GAPDH GAP-Fv 5‟-GACCACAGTCCATGCCATCACT-3‟, Tm, 60C

GAPDH GAP-Rv 5‟-TCCACCACCCTGTTGCTGTAG-3‟, Tm, 60C

66

3

Characterisation of normal and dystrophic

myoblast cell lines

3.1 Introduction

Homeostasis of atrophic/hypertrophic processes is central to the maintenance

of correct mass and functioning in muscle, illustrated by the ability of adult skeletal

muscle to regenerate itself in a manner recapitulating earlier stages of development

(Allbrook, 1981). Attempts to elucidate the intricacies of this system have largely

focussed on a population of muscle resident stem cells; originally identified by Mauro

(1961), based on their anatomical localisation between sarcolemma and basal lamina,

as well as their distinct morphology. These cells are now commonly referred to as

satellite cells; mononuclear cells remaining quiescent in normal adult muscle pending

stimulation to proliferate, whereupon their activated progeny (myoblasts) fuse with

each other or pre-existing myofibres and contribute to the creation/regeneration of

differentiated, multinucleate myofibres. Several satellite cell markers have been

proposed, which delineate quiescent from activated myogenic lineage; the former

include the receptor for hepatocyte growth factor c-Met (Tsukita and Yonemura,

1997), M-cadherin (Bretscher, 1999) and Pax7 (Seale et al., 2000), and the latter

display Myf-5 and MyoD up-regulation prior to S-phase transition followed by

proliferation and continued expression of Myf-5, MyoD and desmin through

subsequent daughter generations (Bretscher, 1999). However, the satellite cell‟s

genetic profile/requirement has recently been proposed to be of age-dependent

67

character, inferring that extrapolations of the embryonic condition to the adult may

hold yet unelucidated complexities (Lepper et al., 2009).

Figure 3.1. Satellite cell activation and division. Illustration depicting the asymmetric and

symmetric division of the satellite cell population of skeletal muscle. Combinations of Pax7,

Myf-5 and MyoD expression (amongst others) distinguish activated satellite cells from the

quiescent progenitor population.

The in vitro culturing of myoblasts (activated satellite cells) has long strived to

recapitulate the in vivo responses of these myogenic progenitors, and derived cultures

have been widely used in research. The most well known perhaps being the

commercially available C2C12 immortalised myoblast line (Schwacke et al., 2009),

which is itself a subclone of the adult C3H crush-injured hind limb-derived C2 cell

line, created as a model of proliferating satellite cells (Yaffe and Saxel, 1977). In the

context of this study, the widely used C2C12 immortalised myoblast line cannot

68

unequivocally be viewed as a true in vitro representation of the in vivo murine satellite

cell; the early work of George Dickson‟s group showed that dystrophin expression is

almost absent in C2C12 myotubes whereas primary cultures of myotubes showed

easily detectable levels (Noursadeghi et al., 1993). However, later work by Betto‟s

group showed that both full length dystrophin and other DAPC complex members are

expressed and easily detectable in both 4 and 8 day C2C12 myotubes (Sandona et al.,

2004), suggesting that clonal differences may exist within populations of

immortalised cells in use throughout the research community. Although widely used

in research, immortalised cell lines carry the perpetual burden of progressive

distancing from the in vivo condition, hence this study utilises both immortalised and

primary myoblast cultures, the source and characterisation of which are presented

here.

A variety of techniques have been proposed for the isolation and in vitro

culturing of myogenic precursor populations derived from the adult muscle of various

animal models (Burton et al., 2000). These protocols have been adapted and

optimised through many years of development by Bischoff‟s group (Bischoff, 1997,

Bischoff, 1990b, Bischoff, 1990a, Bischoff, 1986, Rosenblatt et al., 1995a) in

Washington, who‟s techniques have been subsequently adapted and re-coined by

Partridge‟s group in London (Rosenblatt et al., 1995a) to produce a widely accepted

method of primary rat and mouse satellite cell isolation and culture, largely focussed

on the use of the extensor digitorum longus (EDL) muscle group. Other researchers

have explored the isolation of primary satellite cell cultures from adult murine muscle

using techniques ranging from the differential sedimentation and pre-plating of tissue

slurries (Goodell et al., 2005), as is common when using embryonic or postnatal

tissue, to the cloning of single cell derived colonies from micro-dissected explants

(Merrick et al., 2009). Having trialled several isolation methodologies, an adapted

version of the Rosenblatt method was employed here for primary myoblast culture

establishment (Rosenblatt et al., 1995b).

69

3.2 Myoblast lines

In the absence of human samples, this study aimed to compare ATP responses

between normal and dystrophic murine myoblast cell lines in vitro. Normal and

dystrophic immortalised myoblast cell lines were a gift from Prof. Hans Lochmüller

(University of Newcastle) and represent clonally derived populations, isolated from

mixtures of the TA, GC, Soleus and EDL muscles of 4-12 week male H-2Kb-tsA58

and H-2Kb-tsA58/mdx mice, respectively, according to protocols described by

(Morgan et al., 1994, Springer and Blau, 1997). Genotyping of normal and

dystrophic lines using the PCR based, mdx-amplification-refractory mutation system

(ARMS) assay (Amalfitano and Chamberlain, 1996), confirmed the presence/absence

of wild-type and mdx dystrophin alleles, respectively (Figure 3.2.1).

Figure 3.2.1. Expression of wild-type/mdx dystrophin alleles in immortalised myoblast

cultures. ARMS assay-based RT-PCR demonstrating expression of wild-type and mdx

dystrophin alleles confined to normal and dystrophic lines, respectively. The upper band of

>200 bp is a non-specific amplicon. Schematic below illustrates the technical principle of the

ARMS method.

70

Confirmation of myogenic lineage commitment was attempted by

immunocytochemistry using anti-Myf-5 (Braun et al., 1989) antibody (Merrick et al.,

2009). In both normal and dystrophic immortalised myoblast lines, 100% of cells

stained positively for Myf-5 expression, although contrary to the published work of

Janet Smith‟s group at the University of Birmingham, this antibody (Santa Cruz sc-

302) was deemed non-specific due to its propensity for cytoplasmic as well as nuclear

staining in both normal and dystrophic myoblasts and myotubes. However, under

reduced serum conditions, both normal and dystrophic myoblast lines displayed

morphological characteristics of myogenic precursor cells; fusing to form

differentiated, multinucleated, contractile myotubes, with an associated increased in

desmin expression (Figure Appendix 1) (Tang et al., 2007). Immortalised cultures

proliferated strongly in 20% FBS with a typical population doubling time of 2-3 days

for stock flask cultures. Correct system operation was confirmed through the

withdrawal of -interferon; culturing in proliferation media at 37C in the absence of

-interferon induced the cessation of proliferation within 1-2 days, where-by cells

either came off the plate, or adopted a flattened, disc shape morphology, reminiscent

of a cell having undergone senescence.

Although TA, GC, Soleus and FDB all proved suitable for use with the

primary myoblast isolation protocols employed in this study, this study opted to use

soleus muscles for such procedures due to the presence of distinct tendinous

junctions, which aid the dissection of undamaged myofibres. Normal and dystrophic

primary myoblasts were isolated from soleus muscles of male, 4 month C57BL10 and

mdx mice, respectively, according to a modified version of the protocol described by

(Rosenblatt et al., 1995b). Observations derived over a long period of method

development led to the conclusion that the primary cultures derived using this method

are not homogeneous (as will be discussed later). Collagenase digested, single living

myofibres (both normal and dystrophic) retained numerous intricately associated

mononucleate cells, appearing to occupy locations beneath the basal lamina in the

71

classically defined, satellite cell niche (bright field, phase-contrast derived

observations only – basal lamina integrity not assessed) (Figure 3.2.2; A).

Subsequent to plating, mononuclear cells were observed to migrate off mdx-derived

myofibres in a seemingly contact dependent fashion (Figure 3.2.2; B, C and D).

Figure 3.2.2. 1st generation primary myoblast cultures. Representative brightfield images

illustrating the migration and proliferation of myogenic precursors originating from single

isolated living myofibres (A). Colonies derived from mononuclear cells were observed to

originate from the ends as well as mid-sections of fibres (B-D), and displayed high degrees of

proliferative potential upon removal of the original myofibre (E-F).

72

Satellite cells began to migrate off individually isolated, single living

myofibres following approximately 4 days in culture, after which time cells were

culture in „proliferation medium‟ containing higher concentrations of serum, and cells

were allowed to proliferate to approximately 60% confluency (over a further 10 days)

with regular medium changes. Primary myoblasts isolated in this manner shall from

now on be referred to as „first generation myoblasts‟.

Repeated isolations using the same protocol, but substituting „serum-free‟

growth factors (Knockout Serum Replacement; KSR, Invitrogen) for foetal calf serum

(FCS) facilitated the elucidation of two morphologically distinct populations of

mononuclear, myofibre-resident progentior cells: The first population were observed

to retain a spheroidal architecture, lightly adhering to Matrigel (2mg/ml). This

population readily and consistently differentiated into multinucleate contractile

myotubes (Figure 3.2.3; D), and shall here on be referred to as „second generation

primary myoblasts‟. Myogenic lineage confirmation was achieved by

immunostaining of 2nd

generation primary cultures for desmin the muscle specific

intermediate filament protein, desmin (Figure 3.2.4). Conversely, the other distinct

population of mononuclear cells isolated displayed a more protracted morphology,

adhered both rapidly and tightly to the Matrigel substratum and, importantly, did not

form multinucleate myotubes (Figure 3.2.3; E). Indeed, when approaching

confluency this population rapidly and reproducibly induced the differentiation of

proximal myogenic colonies, an effect which appeared to be, to a degree, contact-

dependent (Figure 3.2.5; lower left panels). This non-myogenic population was

observed to reproducibly give rise to two further morphologically distinct populations

of mononuclear cells: One of a radiated, fibroblast-like appearance, and the other of a

highly vesiculated, swollen cytoarchitecture. Subsequent immunohistochemical

analysis confirmed the latter population to be 100% positive for the lipid stain, Oil

Red O (Figure 3.2.5).

73

Figure 3.2.3. 2nd

generation primary muscle cultures. Brightfield images illustrating two

morphologically distinct populations of mononuclear cells generated from single isolated

living myofibres (A); when isolated, cells of spherical shape - seen in the left hand side

colony in (A), became highly proliferative (B) and consistently and uniquely differentiated

into myotubes in reduced serum media (D). Cells displaying a more elongated morphology as

seen in the colony on the right in (A), were also observed to proliferate rapidly, and

consistently and uniquely differentiated into two different morphologies: a basal layer of

fibroblast-like cells and an upper layer of a different, unidentified cell type (C+E).

74

Figure 3.2.4 Desmin expression in wild-type/mdx-derived 2nd

generation primary muscle

cultures. Brightfield images depicting representative colourimetric staining of desmin

expression over 8 days differentiation of normal- and dystrophic-derived 2nd

generation

primary myoblast cultures, visualised by peroxidase staining (dark pink/purple). Primary

75

antibody was omitted from negative staining controls which represent secondary antibody

staining only.

76

Figure 3.2.5. Adipocyte differentiation in 2nd

generation primary muscle cultures.

Brightfield images illustrating the differentiation of non-myogenic cell fractions of primary

cultures into highly vesiculated mononuclear cells staining positively with lipid stain, Oil Red

O. Upper panels of both genotypes are counterstained with haematoxylin. Lower left panels

of both genotypes clearly display myotube-contact-dependant inhibition of adipocyte

proliferation/migration.

77

3.3 Discussion

Muscle groups of differing developmental lineages have been shown to

contain heterogeneous populations of satellite cells in terms of their proliferation and

differentiation potentials (Ono et al., 2010). Indeed, soleus muscle has been reported

to contain higher numbers of mononuclear cells expressing classic satellite cell

markers than those found in EDL muscle (Schultz et al., 2006), and this heterogeneity

within satellite cell populations also extends to sub-populations within individual

muscle groups (Schultz, 1996, Zammit et al., 2004). It has been shown previously

that satellite numbers present in soleus muscle do not significantly differ between

C57BL10 and mdx in adult animals although satellite cells from mdx soleus muscle

display reduced regenerative capacity compared with those of age-matched C57

animals (Reimann et al., 2000). This is contrary to observations made in this study,

where once isolated, dystrophic myoblasts displayed equal, if not superior

proliferative capacity compared with wild-type myoblasts, although this has not been

quantitated here, and may be dependent on the fibre-type of origin.

In addition to myogenic progenitors, the method development described here

also demonstrated the presence of a population of fibro/adipo-type progenitor cells in

intricate association with isolated myofibres. A similar population has recently been

described in normal skeletal muscles, during the characterisation of a population of

Sca-1+ve

/CD34+ve

fibro/adipogenic progenitors (FAPs), clearly distinct from known

myogenic progenitor lineages. Upon muscle injury these cells have been observed to

undergo proliferation and, when cultured, were found to be a concentrated source of

differentiation signals for primary myoblasts (Joe et al., 2010, Natarajan et al., 2010),

alluding to have a potential role as an interacting partner of myogenic progenitors in

muscle diseases, and a candidate for the non-myogenic component observed during

primary culture development. Characterisation and enumeration of both the levels of

these cells adhering to normal and dystrophic myofibres and the levels of P2X7

78

expression within normal and dystrophic populations of these cells remains ongoing.

Interestingly, another study has described a population of muscle-resident non-

satellite cell-derived myogenic precursors, identifiable by their PW1+ve

/Pax7-ve

,

interstitial localisation (termed PICs) and described as enriched in the Sca-

1+ve

/CD34+ve

cell pool (Mitchell et al., 2010), (marker profile that would also describe

the aforementioned FAP population), which display very different lineage

characteristics in their differentiation. This suggests that the two populations may

potentially be related; perhaps differential signalling at the interstitial matrix-myofibre

interface facilitates the transition from PIC to FAP or vice-versa. However, it should

be noted that PICs have only been studied in neonatal mice to date and their existence

in adult muscle has not been documented. FAP-type cells have been demonstrated in

4 month skeletal muscle (Starkey et al., 2011) where their lineage has been proposed

to be distinct from that of a satellite cell. This is based on the observation that

satellite cells cultured in normal myoblast growth media do not undergo adipogenesis

and those cultured in adipogenic media do accumulate cytoplasmic lipid, but do not

fully undergo adipogenic differentiation (Starkey et al., 2011). These observations

would suggest that the FAP-type cells observed in this study rather than originating

from satellite cells themselves are derived from some unknown precursor cell type

residing on isolated myofibres. The suggestion by Starkey that these adipocyte

forming cells represent a contaminating interstitial population, which can simply be

washed off isolated fibres proved not to be true for the soleus muscle fibres used here;

moreover, these „contaminating‟ cells were found so tightly attached to single isolated

myofibres that they could not be removed by washing, vigorous pipetting or even

through trypsinisation without inducing myofibre death. Starkey‟s proposed

distinction between satellite cell and adipocyte lineage, although most recent in the

literature, is however not unequivocal. Many previous studies have previously

documented satellite cells adopting adipogenic characteristics; by inhibition of Wnt

signalling (Ross et al., 2000) or by growth in adipogenic media (Wada et al., 2002)

79

where increased age has been suggested to be a factor (Taylor-Jones et al., 2002).

Therefore, it can be concluded from the current literature that although myofibre

derived satellite cells and adipocytes are of separate lineage under normal growth

conditions, the potential does exist for this relationship to differ in the dystrophic

environment. The relevance of this point to the dystrophic phenotype, where fibrosis

and fatty infiltration are well-documented patho-histological hallmarks of aging and

degenerating skeletal muscle in humans (Lefaucheur et al., 1995, Pastoret and Sebille,

1995, Delmonico et al., 2009) requires further investigation.

In conclusion, the cell lines used in this study can be considered in vitro

representations of activated satellite cells in vivo. Due to the lack of a specific marker

to label all activated myoblast populations, immortalised and primary myoblast

cultures have been isolated and selectively purified by established pre-plating

techniques, to yield cultures with the potential to uniquely form 100% multinucleate,

contractile myotubes (immortalised myoblast lines were isolated and characterised by

Morten Riso; see Appendix 1, the primary myoblast cultures were isolated and

characterised by the candidate). Non-myogenic adipocyte-generating populations

characterised during primary culture establishment are thought to derive from non-

satellite cell origins and were not present in the purified primary myoblast cultures

described here.

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4

Extracellular ATP hydrolysing potential of

immortalised normal and dystrophic myoblasts

and myotubes

4.1 Introduction

The consensus opinion that dystrophin protein expression is very low or even absent

in undifferentiated myoblasts (Houzelstein et al., 1992, Yeung et al., 2006, Trimarchi

et al., 2006, Scott et al., 1988) is reflected by the fact that the majority of work on

DMD is being conducted in fully differentiated myotubes or myofibres. Myoblasts

are not expected to dysfunction due to the absence of dystrophin. However, contrary

to this view, an increased purinergic sensitivity in dystrophic lymphoblastoid cells has

previously been described (Ferrari et al., 1994); also cells that contain dystrophin

mRNA but have undetectable levels of protein. Moreover, our lab has described a

similar phenotype in immortalised dystrophic myoblasts (Yeung et al., 2006). These

mdx cells do not express protein or full-length dystrophin mRNA due to a premature

stop codon occurring through point mutation at exon 23 (Sicinski et al., 1989) and the

resulting nonsense-mediated decay (Buvoli et al., 2007, Sedlackova et al., 2009).

Purinergic responses in these cells have been shown to be significantly altered when

compared to their normal equivalent (C57BL10) myoblasts, which do express full

length dystrophin mRNA but no detectable protein (Yeung et al., 2006). Importantly,

altered sensitivity to extracellular nucleotides has been demonstrated for P2X and also

P2Y receptors in these cell lines (manuscript in preparation), and this prompts the

question as to whether the effects are specific to altered subunit expression of

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individual purinergic receptors and their splice variants, or whether there is a more

global alteration of nucleotide metabolism in the vicinity of these receptors.

Extracellular ATP can produce various effects acting via P2-purinoceptors and it is

rapidly broken down by ecto-ATPases that limit its effect (Lavoie et al., 2011). Ecto-

ATPases are ubiquitous in eukaryotic cells (Plesner, 1995) and have been shown to be

involved in modulation of P2X receptor responses in muscle (Sneddon et al., 1999).

The expression of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) and

other more atypical ATP-hydrolysing proteins, such as -sarcoglycan has been

documented in muscle (Betto et al., 1999). The -sarcoglycan (SG) protein is a

component of the sarcoglycan complex of dystrophin-associated proteins, all of which

are severely reduced at the sarcolemma in the dystrophic condition (Ohlendieck and

Campbell, 1991, Ohlendieck et al., 1993). SG ecto-ATPase activity has been

characterised in the well studied C2C12 mouse muscle cell line, where in myotubes it

was predicted to provide about 25% of the overall ATP-hydrolysing activity

(Martinello et al., 2011) and therefore play an important role in the modulation of

purinergic receptor signalling (Sandona et al., 2004). The exact functional role of any

of the SG proteins has yet to be established, but it is known that mutations in the

SG gene result in Limb-Girdle Muscular Dystrophy type 2D (LGMD-2D), which

shares a similar phenotype with DMD (Duclos et al., 1998). This lead to the

hypothesis that the abnormalities in purinergic signalling described in dystrophic

myoblasts may result from the inability of these cells to metabolise high [ATP]e (0.25-

4mM), arising from both a release of lots of ATP from damaged dystrophic muscle

fibres (Bodin and Burnstock, 1996) combined with the reduced ecto-ATPase potential

due to loss of SG from its normal extracellular location.

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4.2 Results

In order to test the hypothesis that loss of -SG ATPase activity from the

extracellular face of the DAPC complex in some way confers heightened purinergic

sensitivity to dystrophic myoblasts, ecto-ATPase activity was analysed in

immortalised cultures of normal and dystrophic myoblasts. Initially, RT-PCR

analysis was used to confirm SG mRNA expression in normal and dystrophic

myoblasts (Figure 4.2.1; A). Both cell types showed expression of this transcript

however no estimation of the levels of expression were made as this PCR data cannot

be considered quantitative. Western blot analysis showed that SG protein levels

were below the detection threshold (data not shown). To make a functional

assessment ATP hydrolysing activity has been compared in control and dystrophic

cells. Several methods were trialled for the live-cell quantification of ectoATPase

potential; luciferase based detection methods (Promega) proved too insensitive for the

detection of small changes within a broad [ATP]e, range. Similarly, an ascorbic acid-

based inorganic phosphate detection method (Gawronski and Benson, 2004) was also

tested and actually proved to be the most accurate method of all, yet could not be

scaled up to microplate format due to the lack of the correct 655nm filter wheel in the

plate reader. A useable methodology was devised, adapted from the Malachite green

based inorganic phosphate detection method described by (Sandona et al., 2004),

itself a modification of the assay described by (Lanzetta et al., 1979). The malachite

green-based assay also demanded microplate format absorbance values to be read at

655nm, yet this assay proved more flexible than the ascorbic acid assay, hence the

available 600nm filter provided an acceptable degree of accuracy. No significant

difference was found between the Km or Vmax values of normal and dystrophic

myoblasts (P>0.05; Figure 4.2.1; B and C, respectively), although there appeared to

be a trend towards a reduced Km value in both dystrophic myoblasts and myotubes

compared with their normal counterparts. As expected, significant increases in Vmax

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were observed upon differentiation of the cells into myotubes (P<0.0001; Figure

4.2.1; C) - increases from 0.007 to 0.14 and 0.015 to 0.12 IU/mg of protein were

observed for normal and dystrophic lines, respectively. However, no significant

differences were found between Km or Vmax values relating to normal and dystrophic

4 day myotubes, or indeed between HK2b-tsA58-derived and C2C12 wild-type

myotubes (P>0.05; Figure 4.2.1; C).

Figure 4.2.1. ATP hydrolysing potential of wild-type- and mdx-derived immortalised

myoblast cultures. -Sarcoglycan (-SG) mRNA was detected in both normal and

dystrophic immortalised myoblast lines by RT-PCR (A). Myoblast/myotube ATP

hydrolysing potentials were assessed through determinations of Km (B) and Vmax (C) –

ANOVA revealed no significant differences between normal and dystrophic cells in terms of

either parameter (P>0.05). Significant increases in Vmax were recorded for myotube cultures

(4 days in differentiation media) of both genotypes compared with their respective myoblast

84

cultures. Error bars represent SE, df=4 (C; *P<0.0001). C2C12 cells represent a positive

experimental control (see text).

4.3 Discussion

The proposed functional link between SG and LGMD2D stems from the

observation that nearly all known mutations resulting in this phenotype can be

mapped to the extracellular domain of the SG protein, which has been shown to

demonstrate ecto-ATPase activity in vitro (Betto et al., 1999). Due to the potential

for loss of ecto-ATPases such as SG from the extracellular membrane of dystrophic

cells, it was hypothesised that differences in the [ATP]e hydrolysing potential of

myoblasts could be responsible for the purinergic signalling abnormalities observed

in immortalised dystrophic myoblasts (Yeung et al., 2006). Expression of ecto-

ATPases, such as SG, have previously been characterised in C2C12 myoblasts. It

was suggested to play a role in P2 purinergic signalling through limiting ATP

accessing receptors present on the extracellular membrane of these cells (Sandona et

al., 2004). Importantly, although the assay employed here was not -SG specific,

pharmacological profiles indicative of SG activity were recorded despite the fact

that only SG mRNA and not protein have been detected. Here, immortalised

cultures of normal and dystrophic myoblasts were shown to express SG mRNA

(Figure 4.2.1; A). In agreement with the work of (Sandona et al., 2004) in C2C12, the

immortalised cells used here also displayed an approximately 10-fold increase in ecto-

ATPase activity upon differentiation (Figure 4.2.1; C).

Characterisation of the ATP-hydrolysing potential of normal and dystrophic

myoblast cultures at high [ATP]e was achieved, and kinetic profiles were derived

from dose response curves from five independent experiments comprising triplicate

samples. Non-linear curve fitting (Origin 7.0) showed that no significant alteration in

extracellular nucleotide hydrolysing potential existed between normal and dystrophic

85

myoblasts in terms of Km or Vmax, and moreover that any significant differences in

purinergic signalling response induced through exposure to high [ATP]e (Yeung et al.,

2006) were more likely due to differences in purinergic receptor expression and/or

function at the protein level, potentially rendering the extracellular membrane more

sensitive to purinergic stimulation, rather than due to the agonist degradation

inadequacy that was hypothesised here. However, since the assay employed here was

not specific to any individual ATPase enzyme, the observed trend displaying

decreased Km values in dystrophic versus normal myoblasts and myotubes (Figure

4.2.1; B) could potentially indicate that different ATPase enzymes may be operating

between the two genotypes. It is therefore possible that other ATPases (with

different, in this case lower Km values) are actively compensating for the loss of -SG

in the dystrophic cell line, since the Vmax values appear similar (Figure 4.2.1; C).

Testing this hypothesis would require the purification of -SG enzyme from both

normal and dystrophic myoblasts and myotubes for pharmacological characterisation.

The demonstration of significantly increased Vmax values in both normal and

dystrophic myotube cultures, compared with cultures of the same undifferentiated

cells (Figure 4.2.1; C) served as a positive experimental control, and was in agreement

with the finding that levels of SG expression increase with increasing confluency

and the onset of differentiation (Sandona et al., 2004, Martinello et al., 2011).

Sandona‟s group has previously described an increase in Vmax, from approximately

8.6-4

to 0.04 IU/mg protein, using C2C12 myoblast cells grown under differentiation

conditions for 4 days (Sandona et al., 2004), and more recently provided a revised

value of 0.03 to 0.09 IU/mg protein (Martinello et al., 2011). The changes in Vmax

observed here for immortalised normal and dystrophic myoblasts and myotubes

cultured in the same manner were 0.007 to 0.14 and 0.015 to 0.12 IU/mg of protein,

respectively and thus higher than C2C12 values. For further clarification, Vmax has

been calculated for C2C12 myotubes using the method described here and similarly

86

higher values were obtained. Interestingly, this difference represents an

approximately 3.5 fold increase in Vmax in myotubes derived from the immortalised

cells used here compared with the C2C12 values published by Sandona‟s group in

2004 but only an approximately 2 fold difference compared with the recent data from

the same group (Martinello et al, 2011) for, presumably, the same C2C12 cell line.

The reason for these discrepancies is unknown and possible sources of variability may

be due to different types of spectrophotomeric instrumentation, sources of reagents,

sample incubation times and temperatures, or indeed, cell lines of differing origin.

Km values obtained for these cells could not be compared with Sandona‟s data, as this

group elected to determine their Km values from transfected HEK-293 cells, where

they found Km = 3.71 mM ATP and Vmax = 0.005 IU/mg protein. Km values for the

immortalised normal and dystrophic lines were found to be much lower; 1.04 and

0.68 mM ATP, respectively. Reasons for this again could relate to differences in

curve-fitting approaches used, or possibly that SG displays alternate substrate

specificity dependent on the cell type by which it is expressed. However, close

examination of Sandona‟s group's data does suggest a biphasic dose response, not

observed in the experiments described here. Such a response is uncharacteristic of a

bona fide ecto-ATPase, and would certainly affect the derivation of a true Vmax value.

Indeed, perhaps clarification of Sandona‟s groups claims would be better achieved

through the demonstration of heightened ATP-induced sensitivity in primary

myoblast cultures derived from the SG-deficient mouse model of LGMD2D

(Duclos et al., 1998).

To conclude this chapter, it has been shown here that ectoATPase activity and

therefore ATPe hydrolysing potential, does not significantly differ between normal

and dystrophic myoblasts or myotubes. Should a difference have been found then

further investigations into the component of such an effect specific to a particular

ectoATPase such as -SG would have been further explored. However, the data

87

presented here does not unequivocally exclude the possibility for modulation of the

ATPe hydrolysing reaction in dystrophic cells; the trend (although not statistically

significant) towards lower Km for this reaction in dystrophic versus normal cells may

indicate a compensation by some unidentified ectoATPase which is may be masking

the effect of losing -SG activity from the extracellular face of the sarcolemma.

88

5

P2X7 splice variant analysis in normal and

dystrophic cells and tissues

5.1 Introduction

Nine splice variants of the P2X7 receptor have been cloned in humans (P2X7

„A‟-„J‟) (Cheewatrakoolpong et al., 2005, Feng et al., 2006), the expression

levels/patterns of which have been little explored. The main variant, P2X7A encodes

the classical 595aa subunit with the N-terminal region, first transmembrane domain,

large extracellular loop, second transmembrane domain and the long C-terminal tail.

P2X7B transcript retains the intron separating exons 10 and 11 which generates a

premature stop codon resulting in P2X7 isoform with 249 C-terminal amino acids

following residue 346 replaced by unique 18aa generating a novel C-terminus, as well

as a modified transmembrane domain II.

P2X7B variant has been shown to be ubiquitously expressed in human tissues

with highest levels found in the brain, spinal cord, lymphocytes and lymph nodes.

This receptor variant displays similar responsiveness to ATP and BzATP as the

P2X7A variant, but is deficient in large pore formation and caspase-mediated

apoptotic cascade induction (Cheewatrakoolpong et al., 2005). P2X7B was also

shown to form functional homo- and hetero-oligomeric trimers when co-expressed

with P2X7A in HEK-293 cells, serving to potentiate wild-type P2X7A receptor

responses, and its expression was up-regulated upon mitogenic stimulation in

peripheral blood lymphocytes, with accompanying increases in ER Ca2+

content,

89

NFAT nuclear translocation and cellular ATP content, suggesting a role for this

variant in tumour progression (Adinolfi et al., 2010).

P2X7J, another C-terminally truncated variant (lacking the entire C-terminal

tail, the second transmembrane domain and the last third of the extracellular loop) was

cloned from, and so far has only been shown to be expressed in, cervical epithelial

cancer cells. P2X7J has been shown to be a non-functional receptor variant but able

to negatively regulate the apoptotic cascade-inducing properties of the P2X7A

receptor variant through direct hetero-oligomerisation (Feng et al., 2006). The

subtleties of such an interaction conferring potentially dramatic changes in disease

pathology are currently being explored for therapeutic manipulations, with a

monoclonal antibody against the P2X7J variant pending evaluation for its potential as

a growth inhibitor in cervical neoplasia (Adinolfi et al., 2010). Such developments

highlight both the intricacies of P2X7 signalling and the potential for the abnormal

functioning of this receptor in other pathologies, including muscle disorders.

90

91

Figure 5.1.1 P2X7B and P2X7J splice variant structures. Schematics depicting P2X7B

(A) and P2X7J (B) sequence alignments with associated alterations in receptor structures.

Lower case letters denote residues differing between P2X7A and P2X7B variants -

Transmembrane domain 1 (TM1); yellow, TM2; green, novel C-terminus of P2X7B variant;

red (A). Almost complete loss of exon 8 except for the final base pair (circled) results in

frame shift-generated alternate C-terminus of P2X7J variant, which lacks the second

transmembrane domain and entire C-terminus of the wild-type receptor (B).

Increasing interest in P2X7‟s functional diversity and potential for therapeutic

manipulations have led to investigations into the splicing patterns of this receptor in

mice, where functional relevance in models of disease may be extrapolated more

easily. Specific mouse splice variants have been identified: these include two

transcripts encoding N-terminal isoforms: P2X7a, and P2X7k. P2X7k has a unique

exon 1 encoding specific 42aa of alternative N-terminus and transmembrane 1 domain

(Nicke et al., 2009). The P2X7k‟ variant has been shown to form functional

homotrimers when expressed in xenopus oocytes and HEK-293 cells, and is referred

to as the “killer” variant, so named for its rapid activation and reduced deactivation

kinetics compared with the P2X7a variant. Coupled to its proposed constitutive

dilation and pore formation upon agonist application, the „k‟ variant is deemed more

sensitive to ATP than the „a‟ variant. P2X7k has been shown to be expressed in all

tissues expressing P2X7a.

More recently, two C-terminal variants: P2X7b and P2X7c have been

identified as a result of a collaboration between Dr Murrell-Lagnado, Pharmacology

Department, Cambridge University, The Babraham Institute Bioinformatics and our

laboratory (see Results). Bioinformatic analysis of mouse P2X7 gene structure

followed by searches of Ensembl predictions combined with analysis of expressions

sequence tags (ESTs) over this region in mouse genome identified these two putative

92

alternative 3‟ exons: P2X7b variant encodes an isoform with a significant truncation

of the C-terminal tail, being 164 amino acids shorter than the P2X7a variant (431 &

595aa, respectively). P2X7c cDNA has been cloned as an element of this thesis and

is described in the Results below. Protein sequence alignments comparing all known

P2X7 splice variants indicate that a region of conserved homology exists in exon 2,

proximal to the N-terminal portion of the extracellular loop in all rodent splice

variants as well as the two currently characterised human splice variants; B and J

(Figure 5.1.3). This region has been proposed to contain both sites of ATP binding

(Worthington et al., 2002) and ADP-ribosylation (Adriouch et al., 2008). However,

the same region of homology in the wild-type human P2X7A variant is found in exon

12, proximal to transmembrane domain II (Figure 5.1.3). Conservation of amino acid

sequence homology between P2X7B and P2X7J and rodent P2X7a, k, b, c and d,

could suggest conservation of functional site location between these variants.

However, the reason for the alternate location of this site in the human wild type

P2X7A variant remains unclear, yet the retention of this domain in different exons

may be of functional significance in interpreting species specific responses.

93

Figure 5.1.2 P2X7k, P2X7b and P2X7c splice variant structures. Schematics depicting

P2X7k (A), P2X7b and P2X7c (B) gene structures and protein sequence alignments. P2X7k

exon 1 (1‟) position is indicated in relation to other exons (numbered bars) and start and stop

codons (A; upper panel). Differences in amino acid sequences between P2X7a and P2X7k

are shown; boxes represent similar and identical residues, asterisks represent residues

94

conserved between all rat P2X receptors, underlining denotes predicted TM1 domain (A;

lower panel). The full length P2X7 receptor is encoded by 13 exons with the final exon

encoding the long C-terminus relevant to P2X7b and P2X7c variants. These variants encode

much shorter C-terminal sequences than the P2X7a variant. Variant P2X7b gives rise to a

receptor that is truncated at position 430 and P2X7c has an alternative C-terminus with

additional 11 amino acids beyond the common residue 430 (B; lower panel). The genomic

organization of these two alternative transcripts is depicted in B; upper panel.

95

NP_035157.2| SDKLYQRKEPVISSVHTKVKGIAEVTENVTEGGVTKLGHSIFDT--ADYT 94 a

ACR61396.1| SDKLYQRKEPLISSVHTKVKGVAEVTENVTEGGVTKLVHGIFDT--ADYT 91 k

NP_001033934.1 SDKLYQRKEPVISSVHTKVKGIAEVTENVTEGGVTKLGHSIFDT--ADYT 94 b

NP_001033928.1 SDKLYQRKEPVISSVHTKVKGIAEVTENVTEGGVTKLGHSIFDT--ADYT 94 c

NP_001033976.1 SDKLYQRKEPVISSVHTKVKGIAEVTENVTEGGVTKLGHSIFDT--ADYT 94 d

NP_002553.3| IDFLIDTYSSNCCRSHIYPWCKCCQPCVVNEYYYRKKCESIVEPKPTLKY 400 A

AAX82087.1| SDKLYQRKEPVISSVHTKVKGIAEVKEEIVENGVKKLVHSVFDT--ADYT 94 B

AAX82088.1| SDKLYQRKEPVISSVHTKVKGIAEVKEEIVENGVKKLVHSVFDT--ADYT 94 C

AAX82089.1| IDFLIDTYSSNCCRSHIYPWCKCCQPCVVNEYYYRKKCESIVEPKPTLKY 230 D

AAX82090.1| SDKLYQRKEPVISSVHTKVKGIAEVKEEIVENGVKKLVHSVFDT--ADYT 94 E

AAX82091.1| IDFLIDTYSSNCCRSHIYPWCKCCQPCVVNEYYYRKKCESIVEPKPTLKY 111 F

AAX82092.1| -----------------------------------------MTP--GDHS 7 G

AAX82093.1| IDFLIDTYSSNCCRSHIYPWCKCCQPCVVNEYYYRKKCESIVEPKPTLKY 310 H

ABD59798.1| SDKLYQRKEPVISSVHTKVKGIAEVKEEIVENGVKKLVHSVFDT--ADYT 94 J

. .

NP_035157.2| FPLQGNSFFVMTNYVKSEGQVQTLCPEYPRRGAQCSSDRRCKKGWM---D 141 a

ACR61396.1| LPLQGNSFFVMTNYLKSEGQEQKLCPEYPSRGKQCHSDQGCIKGWM---D 138 k

NP_001033934.1 FPLQGNSFFVMTNYVKSEGQVQTLCPEYPRRGAQCSSDRRCKKGWM---D 141 b

NP_001033928.1 FPLQGNSFFVMTNYVKSEGQVQTLCPEYPRRGAQCSSDRRCKKGWM---D 141 c

NP_001033976.1 FPLQGNSFFVMTNYVKSEGQVQTLCPEDLSN--LQESNQTSKPLLR---N 139 d

NP_002553.3| VSFVDESHIRMVNQQLLGRSLQDVKGQEVPRPAMDFTDLSRLPLALHDTP 450 A

AAX82087.1| FPLQGNSFFVMTNFLKTEGQEQRLCPEYPTRRTLCSSDRGCKKGWM---D 141 B

AAX82088.1| FPLQGNSFFVMTNFLKTEGQEQRLCPE----------------------E 122 C

AAX82089.1| VSFVDESHIRMVNQQLLGRSLQDVKGQEVPRPAMDFTDLSRLPLALHDTP 280 D

AAX82090.1| FPLQGNSFFVMTNFLKTEGQEQRLCPEYPTRRTLCSSDRGCKKGWM---D 141 E

AAX82091.1| VSFVDESHIRMVNQQLLGRSLQDVKGQEVPRPAMDFTDLSRLPLALHDTP 161 F

AAX82092.1| W---GNSFFVMTNFLKTEGQEQRLCPEYPTRRTLCSSDRGCKKGWM---D 51 G

AAX82093.1| VSFVDESHIRMVNQQLLGRSLQDVKGQEVPRPAMDFTDLSRLPLALHDTP 360 H

ABD59798.1| FPLQGNSFFVMTNFLKTEGQEQRLCPEYPTRRTLCSSDRGCKKGWM---D 141 J

.:*.: *.* . * : :

NP_035157.2| PQSKGIQTGRCVPYDKTRKTCEVSAWCPTEEEKEAPRPALLRSAENFTVL 191 a

ACR61396.1| PQSKGIQTGRCIPYDQKRKTCEIFAWCPAEEGKEAPRPALLRSAENFTVL 188 k

NP_001033934.1 PQSKGIQTGRCVPYDKTRKTCEVSAWCPTEEEKEAPRPALLRSAENFTVL 191 b

NP_001033928.1 PQSKGIQTGRCVPYDKTRKTCEVSAWCPTEEEKEAPRPALLRSAENFTVL 191 c

NP_001033976.1 PRAK-------------------------KEEGLSLELA----------- 153 d

NP_002553.3| PIPGQPEEIQLLRKEATPRSRDSPVWCQCGSCLPSQLPESHRCLEELCCR 500 A

AAX82087.1| PQSKGIQTGRCVVHEGNQKTCEVSAWCPIEAVEEAPRPALLNSAENFTVL 191 B

AAX82088.1| FRPEGV-------------------------------------------- 128 C

AAX82089.1| PIPGQPEEIQLLRKEATPRSRDSPVWCQCGSCLPSQLPESHRCLEELCCR 330 D

AAX82090.1| PQSKGIQTGRCVVHEGNQKTCEVSAWCPIEAVEEAPRPALLNSAENFTVL 191 E

AAX82091.1| PIPGQPEEIQLLRKEATPRSRDSPVWCQCGSCLPSQLPESHRCLEELCCR 211 F

AAX82092.1| PQSKGIQTGRCVVHEGNQKTCEVSAWCPIEAVEEAPRPALLNSAENFTVL 101 G

AAX82093.1| PIPGQPEEIQLLRKEATPRSRDSPVWCQCGSCLPSQLPESHRCLEELCCR 410 H

ABD59798.1| PQSKGIQTGRCVVHEGNQKTCEVSAWCPIEAVEEAPRPALLNSAENFTVL 191 J

.

Figure 5.1.3 Multiple protein sequence alignment - Human and rodent P2X7 splice

variants. CustalW2 multiple sequence alignment depicting the singular region of amino acid

sequence homology conserved between all human (uppercase) and rodent (lower case) P2X7

receptor splice variant sequences proteins. In the above section of sequence above „.‟, „:‟

and „*‟ denote low, medium and high level sequence homology, respectively, with 100%

conservation shown in red.

96

All splice variants form functional homotrimers when expressed in HEK-293

cells and xenopus oocytes, demonstrating both calcium channel activity and large pore

opening, yet the kinetics of receptor activation for the three C-terminal variants is

significantly altered in terms of Ca2+

currents; „b‟ and, „c‟ all display <10% of the

BzATP-induced current density observed for the P2X7a variant, and reduced surface

expression when compared with P2X7a in transfected cells. Moreover, the P2X7c

variant actually displays a dominant-negative effect on the observed BzATP induced

current density when co-expressed with the P2X7a variant. The possibility that these

variants may hetero-oligomerise has been confirmed by coimmunoprecipitation pull

down assay in the collaborating laboratory (Dr. Murrell-Lagnado; personal

communication).

The significance of the existence of alternate P2X7 splice variants is

manifested in the two currently available P2X7 knockout mouse lines: the

GlaxoSmithKline (GSK)-generated line, which carries a LacZ transgene insertion in

exon 1 (Solle et al., 2001, Sikora et al., 1999) and the Pfizer-generated line generated

via a neomycin cassette insertion at exon 13 (Solle et al, 2001); The P2X7k variant

escapes inactivation in the GSK P2X7 knockout mouse, where its expression has been

shown to be ubiquitous and highest in spleen (Nicke et al., 2009). The functional

importance of this variant is demonstrated by the increased P2X7 receptor-mediated

T lymphocyte responses in the GSK P2X7 knockout mouse, where specific P2X7

receptor activation and large pore formation coincide with the induction of apoptosis

(Taylor et al., 2009). Subsequent investigations in our laboratory have also rendered

the Pfizer P2X7 knockout mouse a splice variant knockout, where P2X7 alternative

splicing variants „b‟ and „c‟ escape inactivation (unpublished data).

Importantly, these observations may explain the antibody specificity issues

that have long haunted the P2X7 receptor field, where different antibodies generate

spurious staining patterns, some giving positive staining in knockout animals (Sim et

97

al., 2004) and leading to the hypothesised existence of a „P2X7-like‟ protein

possessing similar immunogenicity. An examination of the P2X7 splice variant

expression profile of skeletal muscle represents a novel undertaking and considering

the specific properties of the different variants, it could potentially serve to explain the

previously documented differences in P2X7-mediated pharmacological responses

between normal and dystrophic immortalised myoblast cell lines (Yeung et al., 2006).

5.2 Results

5.2.1 Cloning of a novel C-terminally truncated P2X7c variant

The full length P2X7c transcript encodes a 442aa C-terminally truncated

isoform with a novel 12aa C-terminus (Figure 5.1.2). Using specific primers based on

mRNA sequences obtained from bioinformatic analyses the 1.8kb full length P2X7c

cDNA was amplified from 4 month C57BL10 TA muscle and brain (Figure 5.2.1; A).

The significant differences in amplification efficiency were found to be inherent to the

properties of proof-reading DNA polymerase enzymes from different manufacturers -

Takara‟s „LA‟ enzyme demonstrated far superior performance compared with

Promega‟s „Pfu‟ enzyme (Figure 5.2.1; B). PCR amplicons were cloned into a

standard PCT cloning vector (pGEM-T) using the T/A overhangs. Ligated plasmids

were used to transform competent cells and positive colonies containing the correct

cDNA insert were first identified using blue-white screening and by PCR

amplification in crude bacterial extracts with the specific exon9 and exon13c primers

producing 450bp amplicons. Specific colonies were picked, plasmid extracted and

analysed by PCR (Figure 5.2.1; C) and restriction digestion with Not1 and BamH1.

Expected restriction fragments of 3kb+1.4kb and 3.4kb+953bp were obtained (Figure

5.2.1; D). Cloned products were verified by sequencing (Pubmed entry P2X7 Variant

2; NM_001038839 and supported by ESTs: AK089434, AK144585, BB810482,

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BY544874, BY548370, BY764453) and clones were sent to Dr. Murrell-Lagnado‟s

laboratory (Cambridge) for transfection and characterisation in HEK-293 cells.

Figure 5.2.1. Cloning of full length P2X7c variant cDNA. RT-PCR based amplification of

full length P2X7a and P2X7c variants from total mRNA extracts of C57BL10-derived GC

and brain tissues (A). P2X7c variant expression was lower and more difficult to amplify than

P2X7a variant, although this effect was observed to be to a degree, DNA polymerase enzyme-

dependent (B). Amplification of the specific P2X7 exon9a-exon13b region gave the expected

421bp band size in all of transfected bacterial colonies (C). Samples #1 and #4 were selected

for further analysis, both displaying predicted band sizes following Not1 and BamH1

restriction digestion (D).

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5.2.2 Characterisation of P2X7 splice variants expression in normal and

dystrophic myoblasts and muscle groups

Semi-quantitative mqPCR was developed here as a method of quantitating

RT-PCR data where primers for qPCR were too expensive. The aim here was to use

manual sampling and plotting of PCR time courses to characterise muscle group

specific patterns of P2X7 splice variant transcript up-regulation in mdx compared with

normal age matched controls. The mqPCR technique allowed specific splice variant

expression to be quantitated between different muscle groups, which could be

compared with the more sensitive qPCR technique with primers spanning

P2X7exon1-exon2 boundary, which would detect all P2X7 variants descried here.

Mdx TA muscle demonstrated a uniform and significant 0.94-1.03 fold up-

regulation (Figure 5.2.2; A; *P<0.001) in expression of all variants tested except

P2X7k, although a similar average fold change was also observed for this variant

(0.83). Mdx GC muscle demonstrated the largest significant fold changes in

expression levels (Figure 5.2.2; A; *P<0.0001), again across all variants; from 3.3

fold for P2X7k to 6.9 fold for P2X7c. P2X7b variant showed an average 4.8 fold up-

regulation in mdx GC, although this was not found to be significant (P>0.05). mdx

soleus muscle displayed 1.7, 2.3 and 3.2 fold up-regulations (Figure 5.2.2; A;

*P<0.001) in the expression of P2X7a, k, and c variants, respectively, and although

P2X7b was found to be an average of 2.6 fold up-regulated in dystrophic soleus

muscle, this result was again shown to be not significant (P>0.05). No significant

differences were found in P2X7 splice variant expression levels between wild-type

and dystrophic FDB muscles (P>0.05). Interestingly, mdx diaphragm was the only

muscle type to display significant differences in splice variant expression patterns in

dystrophic vs. wild-type muscle, with a 2.0 fold up-regulation in P2X7a variant

expression only (Figure 5.2.2; A; *P<0.001) and no significant differences in other

variants (P>0.05). Heart muscle results resembled those of FDB muscle, with no

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significant differences in P2X7 splice variant expression found for any P2X7 variants

(P>0.05).

Immortalised dystrophic myoblasts displayed significant up-regulations of 2.9,

1.8 and 3.4 fold for P2X7a, P2X7k and P2X7c variants, respectively (Figure 5.2.2; A;

*P<0.001) – P2X7b variant expression did not significantly differ between wild-type

and dystrophic immortalised myoblasts (P>0.05). Also the 2nd

generation primary

myoblast cultures demonstrated significant up-regulations in expression of 3.6, 2.4,

3.7 and 4.4 fold for P2X7a, P2X7k, P2X7b and P2X7c variants, respectively (Figure

5.2.2; A; *P<0.001).

Tacman primers for qPCR were available spanning P2X7 exon1-exon2

boundary, thus capable of amplifying all P2X7 variants discussed here. The qPCR for

P2X7 showed higher levels of up-regulation in P2X7 expression than were found

using mqPCR - actually demonstrating up to three times the expression levels

described using mqPCR. Significant up-regulations in P2X7a variant expression were

observed in all mdx-derived tissues tested using this more sensitive technique: 3.2,

13.1, 5.7, 2.5, 3.9 and 1.7 fold up-regulations were found in TA, GC, soleus, FDB,

diaphragm and heart of mdx/wild-type, respectively (Figure 5.2.2; B; *P<0.0001).

Indeed, a much higher level of P2X7 expression was observed in dystrophic

immortalised myoblasts using this technique – where a 10.6 fold difference was

observed in mdx/wild-type cells (Figure 5.2.2; B; *P<0.0001). 2nd

generation

primary myoblasts demonstrated a 3.5 fold up-regulation in P2X7 expression in

dystrophic compared to wild-type cells (Figure 5.2.2; B; *P<0.0001), this figure was

more similar to that seen using mqPCR.

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Figure 5.2.2. P2X7 splice variant analysis in cells and tissues. Expression levels of four

mouse P2X7 splice variants: „a‟, „b‟, „c‟ and „k‟ were assessed in mdx- compared with wild-

type-derived muscle groups using semi-quantitative, mqPCR (A) and quantitative, qPCR (B).

Values are shown as fold differences in expression – mdx vs. wild-type, where wild-type=0.

Error bars represent SE, df=2 (*P<0.01-0.0001).

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5.3 Discussion

Characterisation of the novel 5‟ P2X7k or „killer‟ variant, which displays

faster activation and slower deactivation kinetics than the wild-type P2X7a variant

(Nicke et al., 2009), together with a further two functional 3‟ splice variants, P2X7b

and P2X7c, suggested the potential for differential expression of these splice variants

between normal and dystrophic muscle and cells, which may serve to explain the

differences in purinergic responses previously observed between these cells (Yeung et

al., 2006). Indeed, semi-quantitative mqPCR revealed significant up-regulations of

this P2X7 receptor variant in whole muscle-derived RNA extracts of mdx GC and

soleus muscle compared with age matched wild-type controls. The expression

profiles described here for the P2X7k variant in particular, highlight the potential for

dramatically altered purinergic signalling to be inherent in myogenic precursors

present on myofibres of the mdx mouse at 4 months of age. As the only P2X7 splice

variant in mice to be documented in the literature, the demonstration that P2X7k

variant displays higher kinetic potential due to increased agonist sensitivities and

retention (Nicke et al., 2009) suggests that similar, as yet uncharacterised differences

in agonist sensitivities/selectivities may also exist for other variants. Moreover, this

in turn suggests that antagonist profiles may also vary between variants. Antagonists

were not analysed in relation to the P2X7k variant, which is surprising considering the

potentially high therapeutic gains which could be envisaged through the

pharmacological inhibition of such a potentially damaging splice variant. Indeed,

considering the rapidly increasing diversity of P2X7 receptor mediated responses, the

profiling of antagonist selectivities at mouse P2X7 variants would certainly be

beneficial to the wider research community, facilitating more accurate interpretations

of the role of P2X7 receptors in pathological environments.

P2X7 receptors are expressed on many different cell types in muscle,

therefore, perhaps a more elegant future approach would be to employ one of the

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myogenic regulatory factors such as Myf-5 or MyoD, together with immune cell

markers such as F4/80 or CD11b as internal controls alongside GAPDH, thus

allowing additional comparisons relating any observed differences in expression

levels to the number of cells of myogenic/immune cell lineages. Moreover, counts

comparing age-matched mdx- and C57BL10 derived soleus muscles have shown no

significant differences in satellite cell numbers (Reimann et al., 2000), although

heterogeneous populations of satellite cells have been reported in muscles of differing

developmental lineage (Ono et al., 2010, Schultz et al., 2006), rendering comparisons

between P2X7 expression levels in cells and tissues difficult without further

characterisation of primary myoblast cultures. Tissues from 4 month animals were

used here specifically to negate any problems associated with high levels of

infiltrating cells as seen at earlier time points in mdx mice, and no significant

differences in levels of immune cells were found between wild-type- and mdx-derived

whole muscle extracts (see 8.2.1), implying that the observed increases in mdx

compared with wild-type muscles are not the result of higher numbers of infiltrating

immune cells in these tissues. The possibility that immune cells present in mdx

muscle express higher levels of P2X7 receptors than those present in wild-type

muscle cannot be ruled out, also the possibility exists that another un-quantified

population of non-myogenic cells may be responsible for the differences in P2X7

expression levels seen here, however, both these points have been addressed through

the demonstration of significantly up-regulated P2X7 expression levels in both

immortalised and primary myoblast cultures, confirming the assertion that the effect is

myoblast specific and that dystrophic myoblasts of this age display inherently

heightened purinergic sensitivity.

The novel semi-quantitative mqPCR technique employed here proved a

reliable relative quantitative measure, in that the pattern of P2X7a variant up-

regulation observed in mqPCR was similar to that observed using the more sensitive

technique of TaqMan qPCR - of the order GC>soleus~diaphragm>TA. However,

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fold change values found to be < ~2.5 by qPCR, proved too small to detect using the

mqPCR technique, rendering results obtained by mqPCR for FDB and heart as

potentially less significant than they might have otherwise been. In the future,

TaqMan analysis should be performed to answer this question in details.

The finding that all four P2X7 splice variants; „a‟, „b‟, „k‟ and „c‟, all share the

same general pattern of tissue specific up-regulation (Figure 5.2.2; A) is in itself a

novel proposal, the only marked inconsistency here being the lack of significant

P2X7k variant up-regulation in mdx compared to wild-type diaphragm muscle. Such

an observation may be of interest to immunologists, since the mdx diaphragm at this

age would be expected to contain significant numbers of macrophages, where further

characterisation of the potential for differential regulatory mechanisms of splice

variant expression would be very interesting - although the lack of P2X7k variant

specific antibodies would narrow the possibilities here somewhat.

The results presented in this chapter demonstrate significant upregulations in

P2X7 receptor transcript expression in all dystrophic muscle groups tested, offering

potential explanation for the heightened Ca2+

influx response observed in these cells

(Yeung et al., 2006). Chapter 6 will focus on whether the observed increases in P2X7

mRNA levels in the dystrophic myoblasts and muscles in situ result in increased

P2X7 receptor protein expression levels and the possible functional consequences of

this for dystrophic myoblasts. The data presented here indicate for the first time an

apparent association between expression levels of all P2X7 splice variants, which in

dystrophic skeletal muscle appears to be uniformly elevated, with the exception of the

diaphragm where P2X7k variant appears differentially regulated. The mqPCR

technique, although shown here to be a reliable quantitative methodology, did prove

less sensitive than standard Taqman qPCR. Such error was however expected, and

this work should be repeated using specific Taqman probes to confirm the data shown

here.

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6

P2X7 receptor expression and function studies

in normal and dystrophic myoblasts

6.1 Introduction

P2X7 receptors are ATP-gated cation channels, facilitating fast excitatory

responses to extracellular nucleotides and have been widely studied in inflammatory

disorders and cancers. Orthologues have been cloned and characterised from humans

(Surprenant et al., 1996), mice (Chessell et al., 1998b), rats (Rassendren et al., 1997),

dogs (Roman et al., 2009), guinea pigs (Fonfria et al., 2008), and fish (Lopez-

Castejon et al., 2007, Kucenas et al., 2003). Classical agonist sensitivities

synonymous with P2X7 receptor activation differ significantly from other P2X

receptors; EC50 values for ATP at P2X7 receptors are >100 fold higher in mice, rats

and humans [e.g. 2.3M, 1.4M and 5.5M for P2X4 (Chessell et al., 1998a) and

936M, 123M and 780M for P2X7 (Young et al., 2007), respectively]. The ATP

analogue 2‟,3‟-O-(4-benzoyl-benzoyl)ATP (BzATP) is a well documented, specific

agonist of P2X7 receptors displaying greater potency and current densities than ATP

at the mouse orthologue and is less potent than ATP at other P2X receptors (North,

2002). Some significant species differences exist in the kinetics of both agonist and

antagonist pharmacologies (Gever et al., 2006), exemplified by the finding that the

guinea pig receptor orthologue displays reduced selectivity for BzATP over ATP

(Fonfria et al., 2008). Rapid increases in [Ca2+

]i follow agonist application, with

agonist sensitivities of the order BzATP>ATP in mice, rats and humans; EC50 values

for BzATP at mouse, rat and human orthologues expressed in HEK-293 cells are

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around 10 fold lower than those observed for ATP [285M, 3.6M and 52.4M,

respectively (Young et al., 2007, Chessell et al., 1998a)], hence the rat orthologue is

considered more sensitive than that of human or mouse for its native agonist ATP,

and more importantly here, the human receptor is actually considered more sensitive

to ATP than that of the mouse orthologue. Certain antagonists are considered specific

in inhibiting P2X7 receptor responses (Donnelly-Roberts et al., 2009); the most

widely used being Coomassie Brilliant Blue G (CBBG) (at 1-2M), which also

evokes some low level inhibition at P2X4 receptors (North, 2002).

Moreover, sustained stimulation (>10s), or stimulation with high

concentrations of ATP (1-10mM) in all species cells expressing P2X7 receptors

induces the opening of a large non-selective pore, which facilitates membrane

permeability to molecules <900Da. Such permeability was originally characterised in

lymphocytes and since observed in multiple cell types. This event is usually coupled

to membrane „blebbing‟ and phosphatidyl serine translocation to the outer membrane

leaflet (Skaper et al., 2010). The functional mechanisms and relevance of these

seemingly dramatic cellular responses remain to be defined. Yet recently, P2X7a

variant receptor expression has been shown to be up-regulated in the dysferlin

knockout mouse model of LGMD2B, where a role in P2X7-induced, IL-1beta release

and inflammasome activation have been proposed to contribute to the disease (Rawat

et al., 2010). This is a functional effect more synonymous with macrophage

activation (Di Virgilio, 2007) and highlights the potential role of P2X7 in muscle

disease.

Growing commercial interest in developing more specific pharmacological

inhibitors of the P2X7 receptor has facilitated the disentanglement of P2X7 receptor-

mediated responses from those generated by other members of the extended family of

membrane-bound purine receptors. It has also sparked great interest from the wider

research community. However, the P2X7 receptor field itself is fast becoming

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complex. In addition to ATP-induced P2X7 stimulation, NAD has also been shown

to activate P2X7 receptors in certain cell types. The recently explored, NAD-induced

cell death (NICD), occurring via ADP-ribosylation of P2X7 at Arg125 and Arg133

(Laing et al., 2010), has also been shown to play a significant role in cytolysis-related

disorders; In cases such as bacterial infections, endogenous sources of NAD have

been shown to be sufficient to induce both P2X7 activation and NICD (Seman et al.,

2003b, Scheuplein et al., 2009). The proposed mechanism for the NAD- dependent

P2X7 receptor response involves a functional interaction between P2X7 receptors and

one of the members of the ADP-ribosyltransferase (ART) ectoenzyme family. The

best documented in relation to P2X7 responses is the activity of ART2.2 in T

lymphocytes and transfected HEK cells (reviewed in (Schwarz et al., 2009). NAD-

mediated calcium fluxes through the P2X7 receptor channel have been reported

following ART2.2 mediated ADP-ribosylation of residue R125K of the P2X7

receptor. The transfer of an ADP-ribose moiety from NAD+ to P2X7 is proposed to

effect the covalent retention of trimeric ligand binding (Adriouch et al., 2008,

Schwarz et al., 2009), thus lower concentrations of NAD elicit activation of P2X7

receptors compared with those required for ATP-mediated activation (EC50 for

phosphatidylserine exposure being 2M NAD, compared with 100M ATP; Seman et

al., 2003). ATP and NAD+ both exhibit well documented characteristics of

extracellular signalling molecules, and in an environment rich in ATP/NAD+, such as

degenerating muscle, it is not inconceivable that signalling mechanisms relating to

both mediators may be perturbed.

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Figure 6.1. Action of extracellular NAD and ATP on P2X7 receptors. Illustration

depicting the ability of high, concentrations of ATPe to activate P2X7 receptor ion channel

activity. NADe indirectly affects P2X7 receptor function; acting as a substrate of ADP-

ribosyltransferases (ARTs), which transfer an ADP-ribose moiety from NAD to covalently

occupy the ATP binding sites of P2X7 receptor trimers, constitutively activating the ion

channel.

ADP-ribosylation is not the only post-translational modification (PTM) of

P2X7 receptors.; Glycosylation of extracellular domains is also a prevalent and

important PTM, mediating both correct membrane trafficking of receptor subunits

(Young et al., 2007) and the activation of the ERK signalling cascade following

agonist-induced receptor activation (Lenertz et al., 2010). Moreover, tyrosine

phosphorylation at Tyr343 of the intracellular domain has been proposed to be

essential for linking P2X7 receptor activation to cytoskeletal rearrangements and

signalling feedback (Kim et al., 2001), and palmitoylation of four regions of the C-

terminal tail have been linked to the localisation of P2X7 receptors to lipid rafts

(Gonnord et al., 2009).

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Yet another layer of complexity in the study of P2X7 receptor-mediated

responses stems from the existence of genetic polymorphism and alternatively spliced

variants. Single nucleotide polymorphisms or point mutations have been well

documented for P2X7 receptors, conferring a multitude of functional implications:,

such as an N187D mutation; a predicted hyposensitive facilitator of haematopoietic

malignancy (Chong et al., 2010), and an A348T mutation linked to enhanced IL-1

secretion (Stokes et al., 2010). Mutagenesis studies have sought to elucidate residues

of significant functional relevance in transfected HEK-293 cells and oocytes (Young

et al., 2007, Liu et al., 2008, Schwarz et al., 2009) and many studies have indicated

the importance of the C-terminal tail in membrane targeting, large pore formation and

complex formation (Bradley et al., 2010, Lenertz et al., 2009).

One of the most widely documented functional consequences of P2X7

receptor activation in vitro is the induced Thr/Tyr phosphorylation of the mitogen

activated protein kinase (MAPK) family proteins, extracellular signal regulated

kinases 1 and 2 (ERK1/2). The mechanism of this event has been suggested to be

independent of Ca2+

influx (Amstrup and Novak, 2003, da Cruz et al., 2006, Auger et

al., 2005). This interdependence may be cell type specific, as CBBG (by inhibiting

Ca2+

influx) has been shown to inhibit P2X7-induced ERK1/2 phosphorylation in

microglia (Suzuki et al., 2004) but not in osteoblasts (Okumura et al., 2008). Another

possibility is the potential for P2X7 splice variant-dependent regulation of the

ERK1/2 signalling cascade. This signalling could theoretically be of significant

relevance to the dystrophic pathology, having widely documented roles in the

induction of both cellular proliferation and cell death in other systems (Keshet and

Seger, 2010). ERK1/2 activation is itself characteristically biphasic, peaking strongly

at 5-15mins following agonist application, followed by a lower intensity sustained

activation lasting up to 6 hours (Schliess et al., 1996, Kahan et al., 1992, Meloche et

al., 1992). Upon phosphorylation, ERK1/2 proteins migrate to the nucleus via

integrin-mediated cytoskeletal re-organisation, where they promote cellular

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survival/proliferation, facilitating the G1-S phase transition by relaying

phosphorylation status to various substrates within the nucleus (Kahan et al., 1992,

Meloche et al., 1992), such as c-Fos, Jun, and cAMP response element binding

protein (CREB). In addition to the necessity for ERK1/2 phosphorylation and nuclear

translocation during cell proliferation, the known functional repertoire of these

proteins has recently been widened to include a more intricate balance between cell

survival/proliferation and cell death/apoptosis, thought to occur via a coordinated

integration of pro- versus anti-apoptotic stimuli. With obvious functional parallels

between the documented effects of P2X7 activation and ERK1/2 signalling cascade

induction, the P2X7-mediated component of the ERK1/2 phosphorylation response

may in future prove to be a more ubiquitously conserved link between cellular life and

death, making the environment of dystrophic muscle a very interesting arena for the

study of mechanisms pertaining to the regulation of cell proliferation and

differentiation in general. Consequently, therapeutic parallels may arise between

DMD and other circumstances of abnormal ERK1/2 activation.

6.2 Results

6.2.1 C-terminal P2X7 antibody characterisation

This study has assessed P2X7 protein expression using a novel C-terminal

antibody (Synaptic Systems Ltd), which has been extensively characterised by our

laboratory. This antibody recognises a specific 78kDa band in lysates of P2X7a

transfected HEK-293 cells, which is absent from moc-transfected cells. The same

band is also present in brain and TA muscle lysates of mdx mice and and absent from

both currently available P2X7 knockout mice, confirming specificity of the identified

protein. Glaxo and Pfizer knockout animals express P2X7k and P2X7b, c and h,

respectively, although levels are too low to be detectable in Figure 6.2.1; A.

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Moreover, de-glycosylation of protein samples from both transfected HEK-293 cells

and immortalised dystrophic myoblasts results in a characteristic shift from

approximately 78-65kDa (Young et al., 2007), further confirming antibody specificity

(Figure 6.2.1).

Figure 6.2.1. Western blot-based characterisation of C-terminal P2X7 antibody. The C-

terminal P2X7 antibody used in this study demonstrates specific reactivity to the correct size

78kDa band corresponding with the expected size of P2X7 receptor in whole protein lysates

of P2X7a transfected HEK-293 cells. Smaller bands may represent protein degradation

products or partially de-glycosylated receptors. The same size band is recognised in mdx

brain and TA, and absent from both mock-transfected HEK-293 cells and P2X7 knockout

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(KO) tissues (A). Following de-glycosylation with PNGase F the specific P2X7 protein band

recognised in immortalised dystrophic myoblast lysates demonstrated the expected molecular

weight shift to approximately 65Kda (B). Identical band sizes and size shifts on de-

glycosylation were observed in whole protein lysates of 4 month mdx brain, diaphragm and

GC muscles (C).

6.2.2 P2X7 receptor expression and function in immortalised dystrophic

myoblasts

Following the demonstration of no significant difference in ectoATPase

potential between normal and dystrophic myoblasts (Figure 4.2.1), and the subsequent

and novel demonstration of uniformly upregulated P2X7 receptor mRNA transcript

expression in dystrophic versus normal myoblasts (Figure 5.2.2; B), expression levels

and potential functional consequences of P2X7 protein expression levels have been

investigated in normal and dystrophic immortalised myoblast cell lines. Altered

P2X7 receptor protein expression/function could potentially represent the source of

the previously documented abnormal ATPe–induced Ca2+

influx response in

dystrophic myoblasts (Yeung et al., 2006).

Significantly higher levels of P2X7 protein expression were found in

immortalised dystrophic myoblasts compared with their normal counterparts, where

P2X7 expression levels were undetectable using the immunoblot technique (Figure

6.2.2; B). Moreover, in response to high [ATP]e (500M), dystrophic cells displayed

a rapid and sustained increase in ERK1/2 phosphorylation status; from 40-100%

(phospho-p42/p42) activation within 5-10min, an effect mirrored in the response of

P2X7a variant transfected HEK-293 cells, but not observed in mock-transfected

(P2X7–ve) HEK-293 cells, and only very weakly detectable in the wild-type

immortalised myoblasts, where a small increase in ERK1/2 phosphorylation was

observed in the normal immortalised myoblast cells, increasing to approximately 20%

maximal activation within the 15min time course (Figure 6.2.2; B). Dystrophic

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myoblasts however, actually displayed faster kinetics than transfected cells for the

induction of ERK1/2 phosphorylation, achieving maximal activation within <1min,

compared with a more gradual increase of induction in the transfected HEK-293 cells.

The hypersensitive ERK1/2 phosphorylation response seen in the dystrophic

cells was consistent with significantly higher levels of P2X7 receptor expression in

this cell line. ATP induced ERK1/2 phosphorylation in these cells was confirmed as

P2X7 dependent through the use of specific agonists/antagonist combinations: a 10

min pre-incubation with CBBG (1M) was sufficient to completely abolish the ATP-

induced phospho-ERK1/2 response in dystrophic myoblasts. The levels of phospho-

ERK1/2 response in normal and dystrophic myoblasts exposed to the P2X7 receptor

agonist BzATP (300M) mirrored the responses seen using ATP (500M) and there

were also inhibited by 10 min pre-incubation with CBBG (1M) (Figure 6.2.2; D).

As P2X4 receptor expression have previously been demonstrated in the

normal and dystrophic myoblast lines used here (Yeung et al., 2006), ivermectin

(IVM) was employed as a well known positive allosteric modulator of P2X4 mediated

ATP responses (Priel and Silberberg, 2004) However, no significant increase in

ERK1/2 phosphorylation was observed in cultures of dystrophic myoblasts exposed to

ATP (25M) following pre-incubation with 0.25M IVM (Figure 6.2.2; D).

Moreover, ERK1/2 activation was not observed using 2.5M ATP, further confirming

that the more sensitive P2X4 receptor plays minimal if any involvement in the

observed response.

Next, NAD+ was confirmed as a potential mediator of P2X7 signalling in

immortalised dystrophic myoblasts: 2M NAD+ induced a rapid and strong (yet

transient) increase in ERK1/2 phosphorylation in dystrophic cells, whereas the normal

myoblast line was completely unresponsive to NAD stimulation at this concentration.

No induction of ERK1/2 phosphorylation was observed following NAD+ stimulation

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of HEK-293 cells or HEK-293 cells transfected with P2X7a or P2X7k variants

(Figure 6.2.2; E), confirming the requirement for ART enzyme activity during P2X7

receptor stimulation via ADP-ribosylation (Seman et al., 2003a).

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Figure 6.2.2. Functional analysis of P2X7 receptors in immortalised dystrophic

myoblasts. Representative colorimetric immunocytochemistry of P2X7 receptor expression

in immortalised wild-type and dystrophic myoblasts, revealed by peroxidase staining (A).

Representative immunoblots showing relative levels of phospho-p44/42, p44/42 and P2X7

receptor proteins at specific time-points (0-15 min) following activation with ATP (500M).

– Upper panels: moc-transfected HEK-293 cells (left; negative controls) and HEK-293 cells

overexpressing P2X7a receptor variant (right; positive controls), lower panels: wild-type- and

mdx-derived immortalised myoblasts (B). One way ANOVA demonstrated a significant

increase in both the rate and maximal level of phospho-p42 induction in mdx- compared with

wild-type-derived immortalised myoblasts, error bars represent SE, df=2 (C;*P<0.0001).

Immunoblots representative of p42 phosphorylation induction in response to different P2X7

agonist/antagonist combinations: BzATP (300M) and ATP (500M). CBBG panels

represent the effects of both agonists applied after10 min pre- M CBBG

or with CBBG alone (negative control). Ivermectin (IVM; 0.25M) was used to study the

involvement of P2X4 receptors – all panels represent phospho-p42 induction in mdx-derived

immortalised myoblasts, except where indicated (WT=wild-type) (D). Representative

immunoblots of ERK1/2 phosphorylation in response to NAD (2M). A transient NAD-

mediated phosphor-ERK1/2 response was observed in immortalised dystrophic myoblasts

(top left panel), but not in wild-type myoblasts (top right panel; 10min time point, triplicate

samples). NAD had no effect on ERK1/2 phosphorylation in P2X7a-transfected HEK-293

cells (lower right panel) or P2X7 receptor deficient HEK-293 cells (lower left panel) (E).

6.2.3 P2X7 receptor expression in wild-type- and mdx-derived primary

myoblasts

In order to reinforce the physiological relevance the upregulations in P2X7

mRNA and protein expression (Figures 5.2.2; B and 6.2.2; B, respectively) found in

immortalised dystrophic myoblasts and mdx muscles in situ, P2X7 protein levels were

assessed in normal and dystrophic 1st generation primary myoblast cultures.

Immunostaining using anti-P2X7 antibody (Synaptic Systems) was also carried out to

determine the localisation of P2X7 expressing cells on isolated single living

myofibres and derived myoblast cultures in vitro. Anti-P2X7 antibody staining

revealed that primary myoblast cultures stained positive for P2X7 expression, again

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with varying intensity between myoblasts and myotubes, and also with some degree

of variation between individual myoblasts (Figures 6.2.3.2 and 6.2.3.3).

Immunochemical staining was not easily quantifiable, but Western blot analysis of

protein extracts of primary myoblast cultures confirmed a significant up-regulation in

P2X7 protein expression levels in primary dystrophic myoblasts compared with age-

matched controls (Figures 6.2.3.2), which was in agreement with the up-regulated

mRNA levels observed in immortalised and primary cultures as well as muscle tissues

by qPCR (Figure 5.2.2; B).

As has been previously discussed, the classic satellite cell is by no means the

only myogenic precursor of adult skeletal muscle. Preliminary immunohistochemical

localisation of P2X7 receptor expression in cells attached to single isolated living

myofibres suggested that the majority of P2X7 positive cells were Pax7 negative,

although their location relative to the basal lamina or indeed the integrity of the basal

lamina could not be assessed. Immunocytochemical staining of primary myoblast

cultures revealed that cells from both C57 and mdx mice were 100% positive for Myf-

5 and P2X7 expression, though intensities varied amongst cultures and between

myoblasts and newly formed myotubes, which expressed much higher levels of P2X7

expression.

Co-immunolabelling carried out using freshly dissected muscle groups,

collagenase digested to release individual living myofibres, showed that P2X7

receptors were expressed by two populations of mononuclear cells, one positive and

one negative for Pax7 expression.

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Figure 6.2.3.1. P2X7 receptor localisation in muscle fibres ex vivo. Confocal microscope

immunolocalisation: micrographs demonstrating P2X7 expression (red signal) in two distinct

populations of mononuclear cells present on isolated myofibres: one Pax7 (green signal)

positive (A), the other Pax7 negative (B). However another population of P2X7 positive,

Pax7 negative cells was also observed, with morphology that could not clearly/consistently be

described as mononuclear (C+D). Cell nuclei are visualised by Hoest counterstaining (blue

signal).

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Figure 6.2.3.2. P2X7 receptor expression in 1st generation primary myoblasts.

Brightfield images depicting representative colorimetric immunocytochemistry staining of

P2X7 receptor expression in wild-type- (A) and mdx- (B) derived 1st generation primary

myoblast cultures (revealed by peroxidise). Primary antibodies were omitted for negative

staining controls (C). Representative immunoblot and graphical representation of

densitometric analysis demonstrating significantly increased P2X7 receptor protein

expression levels in mdx- compared with wild-type-derived primary myoblasts (D); error bars

represent SE, df=3 (F;*P<0.01).

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Figure 6.2.3.3. P2X7 receptor expression in 2nd

generation primary myoblasts.

Colorimetric immunocytochemistry of primary myoblast cultures showing Myf-5 (upper

panels) and P2X7 (middle panels) staining in wild-type- and mdx-derived 2nd

generation

primary myoblast cultures. Primary antibodies were omitted for negative staining controls

(lower panels), cell nuclei were counterstained with Methyl green (blue).

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6.2.4 Phosphoprotein analysis following P2X7 receptor stimulation in

normal and dystrophic immortalised myoblasts.

Following the demonstration of significantly elevated P2X7 protein expression

levels in dystrophic versus normal myoblasts in vitro, a wide aiming mass-

spectrometry based approach was adopted to address the immediate functional

relevance of the observed differences in Ca2+

influx and ERK1/2 phosphorylation

following P2X7 stimulation in normal and dystrophic immortalised myoblast lines.

Purified phosphoproteins from both cell types - treated or not with ATP, were

separated by charge, iTraq labelled and analysed by „ESI-LC-MS‟ (Agilent 6200

TOF, Agilent Technologies). In ATP-treated dystrophic compared with ATP-treated

normal myoblasts, a total of 1272 unique phosphoproteins were identified. Of which,

phosphorylation status was observed to be significantly higher for 143, and

significantly lower for 121 individual phosphoproteins. In untreated samples, fewer

significant differences were observed; from a total of 1272 uniquely identified

proteins, 32 displayed significantly increased and 23 significantly decreased

phosphorylation status (dystrophic vs. normal). The vast majority of significantly

decreased phosphoproteins in ATP-treated dystrophic vs normal myoblasts involved

proteins with functions relating to transcription, translation or splicing, such as FACT

complex subunit SSRP1, Histone H1.5, Eukaryotic translation initiation factor 5B,

40S ribosomal subunit S6 and Poly(U)-binding-splicing factor PUF60 (2.9, 3.1, 2.5,

2.77 and 2.4 fold down regulated, respectively). In contrast, the majority of

significantly increased levels of protein phosphorylation in ATP treated cells were

seen in cytosolic proteins considered to be involved in a more immediate functional

signalling cascade induction response, such as protein kinase C delta type (PKC),

ezrin, radixin, moesin and annexin A6 (2.7, 2.3, 1.4, 2.0 and 2.3 fold up-regulated,

respectively). Significant ATP-induced phosphorylation effects (mdx/wild-type) on

these and other proteins of particular functional interest are shown in Figure 6.2.4; B.

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Figure 6.2.4. Changes in protein phosphorylation status in normal and dystrophic

immortalised myoblasts in response to ATPe. iQuantitator generated schematic illustrating

positive (green) and negative (red) fold changes in protein phosphorylation status following

treatment with ATP (500M). Large circles represent average values, extended arms linking

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smaller circles represent SE, df=2. Significant and non-significant values are depicted as

opaque and translucent bars respectively, all values shown represent mdx vs. wild-type. Top

30 average fold changes in protein phosphorylation status in ATP-treated (A) and un-treated

(C) cells are shown, plus a selection of other significant differences in phosphorylation status

in ATP-treated cells relating to proteins of specific interest (B).

6.3 Discussion

The demonstration that P2X7 receptor protein expression is significantly up-

regulated in dystrophic myoblasts represents a novel finding. Western blot analysis

demonstrated that this up-regulation coincided with a functional difference in the

P2X7-mediated phospho-ERK1/2 signalling response following stimulation with high

[ATP]e, suggesting that the P2X7 receptor may have a significant functional role in

the dystrophic milieu surrounding degenerating muscle where such high [ATP]e may

be common (Ryten et al., 2004). ERK1/2 are classically associated with cellular

proliferation (Sharma et al., 2003, Cobb et al., 1991, Kim and Choi, 2010) and more

recently, roles for ERK1/2 in mediating cell death, apoptosis, autophagy and

senescence (Cagnol and Chambard, 2010) have been shown. Thus, the balance

between a healthy proliferation and excitotoxic cell-death may depend on the intensity

or duration of the pro- versus anti-apoptotic stimuli transmitted via ERK1/2 (Pearson

et al., 2001). Indeed, it is now thought that constitutive ERK activation is sufficient

to ultimately induce cell death in certain systems (reviewed in (Martin and Pognonec,

2010), although the intricacies of the functional mechanisms mediating this balance

remain to be elucidated and are thought to occur via a coordinated integration of pro-

versus anti-apoptotic stimuli. ERK1/2 proteins mediate the latter through

phosphorylation/post-translational modification of several pro-apoptotic factors, such

as the tumour suppressors FasL and BAD, which ERK/12 proteins inactivate through

interaction with the FOXO and RSK families of transcription factors respectively.

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The pro-apoptotic/autophagic/senescent routes are less well characterised. Here,

ERK1/2 proteins are again thought to co-ordinate the relevant stimuli, but elucidation

of the complete mechanism remains incomplete. P53 activation, DNA damage, ROS,

death activated protein kinase (DAPK) phosphorylation, FasL cross-linking, and Akt

repression, mitochondrial cytochrome c release and caspase-8 activation have all been

implicated in this regard (comprehensively reviewed by (Mebratu and Tesfaigzi,

2009) and (Cagnol and Chambard, 2010).

The novel data demonstrate not only P2X7 induced ERK1/2 activation in

myoblasts but also a specific functional difference in both basal and maximal levels of

ERK1/2 phosphorylation in dystrophic immortalised myoblasts compared with their

normal counterparts, suggests a possible route for therapeutic intervention in

dystrophic muscle. However, the complexities of such mechanisms are exemplified

by the observation that small molecule activators of ERK1/2 such as Resveratrol,

appear to exert their effects in a cell/tissue specific manner, while reducing the

invasiveness of some tumour types through apoptosis (Maher et al., 2011, Lin et al.,

2011), yet suppressing chemically induced apoptotic cell death in other tumour types

(reviewed in (Gupta et al., 2011) and actually inducing a protective effect in

Huntington‟s disease (Kim and Choi, 2010). Hence, any pharmacological strategy

targeting the ERK1/2 mediated component of muscle cell death in DMD would have

to carefully consider the particular target component and its effect on other aspects of

cellular viability. From the currently available literature, inhibitors of Death

Associated Protein Complex (DAPK) phosphorylation/cytoplasmic accumulation

would be potential candidates (Mebratu and Tesfaigzi, 2009). However, whether

DAPK phosphorylation/accumulation is up regulated in dystrophic muscle or muscle

in vivo is unknown. ATPe induced apoptosis was assayed for in immortalised normal

and dystrophic myoblasts prior to primary cultures becoming available – as predicted,

immortalised lines proved un-responsive to staurosporine or ATPe induced apoptosis

(Figure Appendix 2).

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Functional hetero-oligomerisation has been documented between P2X7/P2X4

receptors following their co-expression in HEK-293 cells (Guo et al., 2007) and also

as endogenous receptors in macrophages (Boumechache et al., 2009), and P2X4 has

also been shown to induce phosphorylation of ERK1/2 proteins (Inoue, 2006).

Therefore, dystrophic myoblasts were co-stimulated with ATP and IVM (as a positive

allosteric modulator of P2X4) to elucidate any P2X4-mediated component of the

purinergic response observed in these cells; low micro-molar concentrations of IVM

(2.5M), have been shown to increase P2X4-mediated Ca2+

influx by up to 10 fold

(Priel and Silberberg, 2004, Khakh et al., 1999). Although low levels of ERK1/2

phosphorylation were induced by low [ATP]e [25M; sufficient to fully activate

P2X4 (EC50, 2.3M; (Jones et al., 2000), but not P2X7 receptors (EC50, 936M;

(Young et al., 2007)] in immortalised dystrophic myoblasts, the magnitude of this

response was approximately 5 fold lower than observed using higher concentrations

(500M), and the same cells proved unresponsive at lower concentrations of ATP

(2.5M) (data not shown). Moreover, the lack of a significant increase in Ca2+

influx

(data not shown) or ERK1/2 phosphorylation in response to co-stimulation with ATP

(25M) and IVM (0.25M) in our cells suggests that although a P2X4-mediated

response may be present, it can be considered minimal in comparison to the P2X7-

mediated response in these cells.

P2X7a and P2X7k variants have recently been shown to differ in their

sensitivities to ADP-ribosylation; low micromolar concentrations of extracellular

NAD trigger P2X7-dependent calcium influx and large pore formation in P2X7k

variant transfected HEK-293 cells, but not cells transfected with P2X7a variant

(unpublished data from Murrell-Lagnado, Cambridge). This difference in sensitivity

has been shown in HEK-293 cells co-transfected with P2X7k and ART2.2, as these

cells are deficient in the expression of ADP-ribosyltransferase enzymes (Schwarz et

al., 2009). ART1 has been shown to be the predominant ADP-ribosyltransferase

126

variant expressed in skeletal muscle, (being originally cloned from rabbit muscle;

(Zolkiewska et al., 1992), and although ART1 and ART2 genes are both expressed in

mouse and human skeletal muscle (Glowacki et al., 2002), only ART1 expression has

been shown to be expressed in cells of myogenic lineage, and only in vitro, where it

proved undetectable in C2C12 and C3H-10T myoblast lines, yet was strongly up-

regulated upon the induction of differentiation in the same cell lines (Zolkiewska et

al., 1992, Friedrich et al., 2008). Considering the significant difference in P2X7k

variant expression levels between normal and dystrophic immortalised and primary

myoblast lines, and the NAD-induced ERK1/2 phosphorylation response specific to

the dystrophic myoblast cell line it would seem a logical progression to quantitate any

differences that might exist between the levels of ART1 expression in these cell lines.

Direct measurements of ADP-ribosylation levels of P2X7 receptors in these cells

could be obtained through treatment with etheno-NAD (a covalent antagonist of

NAD-mediated ADP-ribosylation), followed by immunoprecipitation for P2X7 and

immunoblotting using an etheno-adenosine specific antibody (Krebs et al., 2003,

Friedrich et al., 2008).

Having demonstrated an abnormally high level of functional P2X7 expression

in the immortalised and primary dystrophic myoblast cell lines by qPCR (Figure

5.2.2; B), this study has confirmed the potential for a specific purinergic phenotype to

be operating in dystrophic muscle by demonstrating significant up-regulations in

P2X7 receptor protein levels in both immortalised and primary dystrophic myoblasts

(Figures 6.2.2 and 6.2.3.2, respectively). Results presented here not only report

abnormalities in P2X7 receptor expression between normal and dystrophic myoblasts,

but also question the findings of previous studies proposing a sovereign role for P2X5

in muscle development and myoblast differentiation (Ryten et al., 2002, Ryten et al.,

2004). ATP induced ERK1/2 signalling has previously been demonstrated in

myoblasts (Bennett and Tonks, 1997, Jones et al., 2001) and proposed to be P2X

receptor dependent (Banachewicz et al., 2005). Burnstock‟s group have proposed that

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P2X5 is most salient in this regard, and a role in myoblast differentiation has been

proposed (Ryten et al., 2002, Ryten et al., 2004). Surprisingly, this work has never

been confirmed or extended, and indeed, several inconsistencies are present in the

results presented by both Banachewicz and Burnstock: Firstly, the proposal by

Banachewicz that C2C12 myoblasts display a specific Ca2+

dependent, P2X5-

mediated response is unconvincing given the concentration of ATP (100M) required

to induce the documented effect, which is by far more reminiscent of a P2X7 than a

P2X5 mediated response. Moreover, the ATP induced Ca2+

influx response they

reported was actually activated by the specific P2X7 agonist BzATP (100M) alone.

Burnstock also chooses to use uncharacteristically high concentrations of ATP

(100M) for the induction of the proposed P2X5-mediated phosphoERK1/2 response,

yet lower concentrations of ATP (10M) were used in examining the more sensitive

Ca2+

influx response. Moreover, ERK1/2 activation is itself not normally associated

with P2X5 receptor activation and has only been well characterised following P2X4

and P2X7 stimulation. Therefore, it is unclear why these studies chose to ignore the

potential involvement of P2X7 receptors, despite detecting its transcript in myoblasts

(Banachewicz et al., 2005, Ryten et al., 2004) - it may be because the initial

publications relating to P2X receptor involvement in muscle development focussed on

P2X5 and P2X6 receptors (Meyer et al., 1999); In the light of the current study, a

logical progression would be to characterise P2X7 receptor expression patterns in

developing muscle. However, due to continuing difficulties with antibody specificity

in immunohistochemical staining and the rapid advancements in the development of

more specific P2X7 antagonists, investigation into the role of P2X7 in development

and myoblast proliferation/differentiation would be better conducted using

agonist/antagonist manipulation of specific cellular events. Importantly, P2X7 up-

regulation in mdx-derived primary myotubes has recently been documented (Rawat et

al., 2010), alluding to the potential for a similar mechanism to be potentiating the

pathology of DMD muscles and their mononuclear precursors.

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As previously mentioned, a broadly aimed phosphoprotein characterisation

was attempted using a mass spectrometry-based approach in an attempt to elucidate

the immediate downstream functional consequences of P2X7 receptor activation in

these cells. The rationale for this focussed on the possibility of elucidating other

components of the P2X7-ERK1/2 signalling cascade. Maximal fold changes of ~4

were detected using this approach, which is in keeping with similar studies in the

literature (Mayya and Han, 2009). The effect of charge bias affecting the mass

spectrometry sample column seems more plausible, given the large bias in protein

identification towards that of nuclear proteins. Yet this does not explain the

identification of the ERM proteins being significantly (>1.5 fold) up regulated in

dystrophic cells, unless the degree of phosphorylation of these proteins is large

enough to remain undimmed by the issues of sensitivity. However, phosphoproteins

are characteristically troublesome with regard to mass spectrometry due to their

potential for multi-phosphorylation (ERK1/2 are dually phosphorylated) and their

existence in low abundance of total protein samples. Hence, with the advent of more

specific methodologies, perhaps a phosphoprotein based assessment of particular

groups of related phosphate modifications, or use of more permanent modification

techniques, such as Stable Isotope Labelling of cells (SILAC) (reviewed in (Mayya

and Han, 2009) should be attempted. Moreover, full clarification of the significance

of the “hits” would require Western blot based detection of these phosphoproteins

detected by mass spectrometry and comparison of their phosphorylation status with

that of the ERK1/2 proteins.

Of particular interest in the obtained phosphoprotein data are the significant

up-regulations in phosphorylation status of the ERM proteins ezrin, radixin and

moesin (Tsukita and Yonemura, 1997, Takeuchi et al., 1994, Bretscher, 1999). ERM

represents a well characterised actin binding complex of proteins which has been

implicated in the determination of cell shape, migration and proliferation (Denker et

al., 2000, Ivetic and Ridley, 2004). Protein phosphorylations observed for ATP-

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treated dystrophic vs normal myoblasts suggest that this protein complex may offer

potential avenues for investigating some other rapid ATPe–induced responses

observed in these cells - specifically that dystrophic cells detached from adherent

surfaces within minutes of the addition of high [ATP]e (>5mM). The ERM proteins

link integral membrane proteins to the actin cytoskeleton (Ivetic et al., 2004) and have

been localised to focal adhesions and lamellopodia. Upon phosphorylation, ERM

proteins undergo structural changes induced by relief of intramolecular interactions,

generating an active conformation. In their active conformation, ERM proteins have

been shown to bind actin through their carboxy-terminus and through their amino-

terminus, to bind cell adhesion molecules (Integrin 2; (Tang et al., 2007), scaffolding

molecules (EBP50; (Fouassier et al., 2000), and signalling transducers (Rho GDI;

(Mammoto et al., 2000). The localisation of ERM proteins to focal adhesions and

lamellopodia suggests that they may play a role in the ATP-mediated cellular

detachment observed on exposure of dystrophic myoblasts to high [ATP]e (Yeung et

al., 2006).

Another conspicuous phosphoprotein of potential significance is PKC -

generally regarded as an inducer of cell cycle arrest or differentiation induction,

PKC activation has also been linked to pro-mitotic stimuli, of particular interest here,

is that mediated by ERK cascade induction, although such effects are very much cell

type specific (Steinberg, 2004). Annexin A6 may also be of relevance in this context;

a calcium dependent membrane protein expressed in skeletal muscle, where

functional interactions with PKC have been proposed to act as a negative effector of

MAPK signalling and calcium channel function by Ca2+ induced translocation of

annexin A6 to the plasma membrane (Grewal et al., 2005, Grewal et al., 2010).

Cofilin and destrin have been highlighted due to their proposed roles as actin

depolymerisation factors (Liu et al., 2007) with documented roles in skeletal muscle

in relating to potential abnormalities in actin depolymerisation in myopathies

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(Papalouka et al., 2009). Specific functions for these phosphoproteins in myoblasts

have not been documented, although cofilin has previously been referred to as the

generic “steering wheel” of the cell, defining cell motility (Ghosh et al., 2004).

Nestin, a well known intermediate filament protein and stem cell marker has recently

been ascribed a phosphorylation-dependent role in neuromuscular junction formation

(Yang et al., 2011), and although nestin functions in mononuclear myogenic

precursors are less clearly defined, a role in differentiation has been suggested (Cui et

al., 2009, Kitai et al., 2010).

Interestingly, the actin binding protein zyxin was found to possess a

significantly higher basal phosphorylation status in dystrophic compared to wild-type

myoblasts (untreated). Zyxin phosphorylation has itself been linked to reduced cell-

cell and cell-substrate adhesion through increased LIM domain availability (Call et

al., 2011) in a similar manner to that involved in ERM protein activation.

In conclusion, this chapter presents the results of a mass spectrometry-based

investigation into potential downstream consequences of P2X7 receptor activation in

immortalised dystrophic myoblasts in vitro. Although the data have been generated

following treatment with ATP rather than the more specific P2X7 agonist BzATP,

sufficiently interesting „hits‟ have been generated, which warrant further investigation

to determine their role in the P2X7 specific response to ATPe. In particular annexin

A6, who‟s membrane associated involvement in the IL-1 response in muscle may

parallel that seen in macrophages (Grewal et al., 2010), which would be a novel

finding and of potential therapeutic relevance to DMD pathology.

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7

Effect of micro-dystrophin gene transfection on

P2X7 receptor responses in immortalised

dystrophic myoblasts.

7.1 Introduction

The issue of delivering the large (14kb) full length dystrophin cDNA to effect

artificial expression of partially functional truncated dystrophin proteins has been

addressed in two different ways; through the continued development of larger

capacity vectors and the use of truncated dystrophin transcripts retaining the required

functional elements. Reductions in transgene size have been facilitated following the

discovery that large sections of the dystrophin transcript can be removed or altered

without significant effect on its function. Several studies have sought to assess the

impact of various dystrophin truncations; N-terminal deletions resulting in a BMD

phenotype when expressed in the mdx mouse have been described (Corrado et al.,

1996). It has been demonstrated that the C-terminal region is not required for the

assembly of the DGC (Crawford et al., 2000) and that a truncated variant found in a

BMD patient, lacking large parts of the central rod-domain retained most of its

function (England et al., 1990). This work was subsequently extended to show that

the presence and configuration of certain segments of the rod domain were required

for functionality to be retained (Harper et al., 2002). The cysteine rich domain of

dystrophin is thought to be crucial for functional retention of the DGC complex

(Rafael et al., 1996), as demonstrated by DMD patients presenting with a single base

pair deletion in this region (Tsukamoto et al., 1994). On the other hand, cDNA

132

constructs encoding the small, N-terminally truncated isoforms of dystrophin, e.g.

Dp71 not only failed to improve the dystrophic pathology but had a detrimental effect

(Greenberg et al., 1994, Cox et al., 1994). Therefore, therapeutic attempts to deliver

dystrophin mRNA focus on internally truncated mini- or micro-dytsrophin constructs

lacking the majority of the central rod domain, only retaining the functional N and C

termini, WW, EF and ZZ domains. Such constructs transfected into dystrophic

progenitor cells have been widely used in attempts to revert the dystrophic pathology

through in vivo/in vitro expansion of the dystrophin positive myofibre population

(Acsadi et al., 1991, Vincent et al., 1993, Yang et al., 1998, Rodino-Klapac et al.,

2010, Rodino-Klapac et al., 2011). In the context of P2X7 mediated signalling,

myoblasts transfected with such constructs have been employed by our lab in order to

elucidate the specific effect of dystrophin on P2X7 expression and signalling.

Specific mini-dystrophin cDNAs and immortalised dystrophic myoblasts stably

expressing these constructs were a gift from Dr Rolf Stucka (Friedrich Bauer Institute,

Munich, Germany).

7.2 Results

7.2.1 Characterisation of micro-dystrophin expressing clones

Four micro-dystrophin transduced immortalised myoblast lines were available

for analysis: clones # C8; containing 3759bp cDNA, 25-14a; 3435bp, 125-63; 3705bp

and 24W57; 3696bp. Predicted molecular weights of resultant mini-dystrophin

proteins were: C8: 127kDa, 25-14a; 115kDa, 125-63 and 24W57 both 125kDa

(Figure 7.2.1; A). These established cell lines were characterised for the expression

of their mini-gene constructs by PCR using primers specific for regions spanning

those lost in truncated mini-dystrophin transcripts. PCR products were confirmed as

specific to their region of interest, being absent from both the normal and dystrophic

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immortalised un-transfected myoblasts, where the primers are separated by ~9kb of

extra sequence that was removed when creating the mini-dystrophin transfectants.

Multiple experiments demonstrated the C8 clone to be consistently positive for the

presence of the construct in the genomic DNA (Figure 7.2.1; B) and for expression of

mRNA (Figure 7.2.1; C) and the 25-14a and 125-63 clones both demonstrated lower

levels of expression of their respective transgenes (Figure 7.2.1; B and 7.2.1; C,

respectively) while the 24W57 clone was consistently negative for the presence and

expression of the transgene.

Restriction digests were carried out to further confirm specificity of the PCR

products amplified in the three positive clones: Sac1 digestion produced restriction

products of the expected sizes (Figure 7.2.1; D). Both BamH1 and Xho1 produced

the expected restriction products for the C8 clone but did not cut amplicons from the

25-14a clone, whereas BssS1 produced the expected band sizes in both the C8 and 25-

14a clones. This is consistent with the sequence data, where the 25-14a is 324bp

shorter than the C8 clone, and both BamH1 and Xho1 had predicted restriction sites

within 200bp downstream of the deletion point, whereas BssS1, Sac1 and Hind3 all

have their predicted restriction sites over 400bp downstream (and hence outside) of

the deleted region (based on a restriction map generated from the known sequence of

the 24W57 clone).

The clone expressing the largest mini-dystrophin gene; C8 was selected for

further analysis. Interestingly, attempts to show transgene protein expression by

immunoblotting using a C-terminal anti-dystrophin antibody to detect the transgene

product in total protein lysates from this cell line proved not reproducible, whereas the

same construct transiently expressed in HEK cells gave the expected size protein

product (data not shown). Immortalised dystrophic myoblasts stably transfected with

an empty plasmid vector were used as controls in all experiments.

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Figure 7.2.1. Characterisation of micro-dystrophin transfected H2Kb-tsA58/mdx-

derived myoblasts. Transgene expression levels were assessed in four stably transfected

immortalised dystrophic myoblast lines containing slightly different, CMV promoter driven,

micro-dystrophin constructs (A). PCR of specific micro-dystrophin gene region in gDNA (B)

and RT-PCR of the same region demonstrated that three of the four clones expressed their

transgene, albeit at different levels, with 24W57 being consistently negative for expression

(C). Amplification product specificity was further confirmed by restriction enzyme digestion

using Sac1. Restriction fragments of the expected sizes were obtained in all three of the four

clones expressing detectable levels of transgene cDNA (D). Figure panel (A) adapted from a

diagram kindly provided by Prof. Hans Lochmüller.

135

7.2.2 P2X7 expression and function studies in the C8 cells

The demonstration of abnormally elevated P2X7 receptor expression levels in

dystrophic versus normal myoblasts and muscles in situ invites questions as to

whether the phenomenon is functionally linked to the dystrophic phenotype, i.e. does

dystrophin expression functionally affect P2X7 expression in the mdx mouse model of

DMD? P2X7 protein expression levels were observed to be effectively normalised in

the C8 micro-dystrophin transduced immortalised dystrophic myoblast cell line.

P2X7 receptor expression was virtually undetectable in protein lysates from the mini-

gene expressing dystrophic immortalised myoblast, a profile undistinguishable from

the wild-type cells. At the same time immunoblotting using the same anti-P2X7

antibody demonstrated a significantly higher level of P2X7 receptor protein in the

control- (empty vector transfected) dystrophic myoblasts. Thus the empty vector

transfected dystrophic myoblasts displayed characteristics of the un-transfected

dystrophic line (Figure 7.2.2; A). In the next step, immunoblot analysis using anti-

ERK1/2 and anti-phospho-ERK1/2 antibodies revealed that the normalisation of

P2X7 receptor expression levels in the C8 cell line coincided with the significant

reduction of ATP induced ERK1/2 phosphorylation in the mini-dystrophin

transfected cells, bringing it to levels more reminiscent of the wild-type myoblast line

(Figure 7.2.2; B).

To further analyse the efficacy of various mini-dystrophins and variable

expression levels, all stable transfectants were tested by a colleague for Ca2+

influx

following ATP stimulation. This analysis demonstrated clear differences in response

with the strongest Ca2+

influx effect in C8 cells and no normalisation in 24W57 cells,

which were shown to have no detectable mini-gene expression (Wojtek Brutkowski;

data not shown).

Further proof for the role of dystrophin in maintaining a proper P2X7 function

was sought through shRNA mediated knock-down of dystrophin in wild type

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immortalised myoblasts. However, despite using a commercial system consisting of

lentivirus expressing shRNA, multiple attempts and modifications suggested by the

manufacturer were unsuccessful and dystrophin mRNA levels remained unchanged in

transduced normal myoblast cells (data not shown).

Figure 7.2.2. Normalisation of P2X7 expression and signalling in micro-dystrophin

transfected H2Kb-tsA58/mdx-derived myoblasts. Representative immunoblots showing

normalised levels of P2X7 receptor protein in H2Kb-tsA58/mdx immortalised myoblasts

stably expressing the C8 micro-dystrophin construct (A). The effect of micro-dystrophin

transgene expression also extended to the normalisation of the ATPe-induced phospho-

ERK1/2 response in these cells (B). mdxC8

and mdxev

denote H2Kb-tsA58/mdx myoblasts

containing C8 micro-dystrophin and empty vector, respectively. All immunoblots represent

triplicate experiments.

137

7.3 Discussion

The finding that the expression of a functional, albeit truncated, dystrophin

mini-gene in an immortalised dystrophic myoblast cell line effectively reverted the

hypersensitive purinergic phenotype of these cells to that of their normal counterparts

is intriguing. PCR based analysis convincingly showed that the mini-gene construct

had been stably inserted and was being expressed. It is possible that very low levels

of dystrophin located in specific sub-membrane domains or even dystrophin mRNA

directly are involved in some unknown regulatory mechanism in these cells. The first

indication that dystrophin expression could be linked to P2 receptor function was the

work of (Ferrari et al., 1994) who provided evidence that lymphoblastoid cells from

DMD patients displayed a purinergic phenotype which rendered them highly sensitive

to large increases in [Ca2+

]i in response to high [ATP]e. The important role for

dystrophin mRNA and/or protein in myoblasts is again eluded to by the recent finding

of metabolic abnormalities in dystrophic cells (Onopiuk et al., 2009) and by

abnormally rapid differentiation of isolated dystrophic myoblasts in vitro (Yablonka-

Rouveimi et al). The finding that P2X7 receptor protein expression is up-regulated in

dystrophic myoblasts has parallels with previous studies; lymphoblastoid cells and

myoblasts both express very low levels of dystrophin protein, yet the effect on the

purinergic phenotype in both cell types is profound, suggesting the possibility for

dystrophin protein or mRNA co-ordination/involvement in P2 receptor

expression/recruitment and also potentially, for an important role for dystrophin in

myoblasts.

Dystrophin isoform expression has been shown to be intricately regulated in

both developing/regenerating and mature muscle tissue; Dp71, the shortest of the

dystrophin isoforms comprising a unique seven amino acid N-terminal sequence

followed by only the cysteine rich and C-terminal domains of dystrophin (Bar et al.,

1990) has been shown to be ubiquitously expressed but excluding skeletal muscles,

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where its expression is strictly confined to myoblasts (Rapaport et al., 1992). The

regulatory role of MyoD expression on the efficacy of Sp1/3 transcription factor

binding to Sp boxes upstream of the Dp71 explains Dp71 promoter down-regulation

upon the induction of myoblast differentiation (de Leon et al., 2005). Regulation of

Dp427, the full length dystrophin gene product transcribed from the M-type muscle-

specific promoter, is achieved through the competitive binding of the zinc finger

nuclear factor YY1 to CArG elements recognised by dystrophin promoter bending

factor (DPBF) DNA binding protein (Galvagni et al., 1998) and this mRNA is mostly

expressed upon myogenic differentiation and hence this isoform is the predominant

variant found in mature myofibres (Lev et al., 1987). Interestingly, Dp71 is expressed

in mdx myoblasts but it does not prevent the metabolic and purinergic abnormalities.

The mini-gene transduced cells used in this study utilised cDNA dystrophin isoforms

truncated through deletion of large sections of the central rod domain but containing

the N-, cystein-rich and C-terminal domain of the full-length isoform. It appears,

therefore that it is the 5‟-end or the N-terminal domain of the full-length transcript

that may be essential for the maintenance of normal ATP responses in myoblasts.

Surprisingly, following cell transfection, mini-dystrophin protein was not detectable

in cellular lysates. Possible explanations for this could include low expression due to

rapid promoter methylation, micro-RNA control mechanisms, premature mRNA

degradation through immediate ubiquitination of the mRNA molecules, or the

existence of an RNA storage mechanism for specific 5‟-end containing sequences in

these cells.

The full length dystrophin transcript requires ~16 hours to be transcribed and

is co-transcriptionally spliced (Tennyson et al., 1995), so it would not be un-

reasonable to suppose that this process may begin in the myoblast some time prior to

differentiation in order to optimise translation times upon differentiation. Indeed,

perinuclear nuclear distributions of multiple DAPC complex components including

dystrophin have been documented in undifferentiated human myoblasts, with

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cytoplasmic localisation evident after 4 days of differentiation (Trimarchi et al.,

2006). The storage of DAPC complex members in perinuclear locations prior to the

onset of differentiation implies distinct functional relevance for other cellular

pathways – for any storage mechanism must be monitored and regulated, thus it is not

inconceivable that in the absence of dystrophin, such pathways would never reach a

state of readiness, and any overriding inductions of differentiation would e

accompanied by inappropriate signalling check point controls, potentially, in some

way involving P2X7-mediated signalling cascades. However, in conclusion,

insufficient literature is currently available to conclusively support a functional link

between Dp427 and P2X7 expression, although the experiments using the C8 clone

described here suggest some kind of mechanistic link.

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8

Analysis of P2X7 receptor protein expression in

4 month wild-type and mdx muscle groups

8.1 Introduction

ATP has been known since 1983 to possess extracellular transmitter-like

properties when Kolb & Wakelam demonstrated such action in 12-day cultures of

chick derived myotubes (Kolb and Wakelam, 1983). ATP-induced cation influx in

skeletal muscle was demonstrated soon after by (Haggblad et al., 1985) in similar

chick derived myotube cultures. A role in the developmental initiation of myoblast

fusion has been assigned to both P2X5 and P2X6 receptors who‟s expression has been

shown to disappear immediately prior to myotubes formation in developing chick and

rat muscle (Meyer et al., 1999, Ryten et al., 2001, Ryten et al., 2002). The same

group also conducted the first analysis of P2 receptors in mdx skeletal muscle, finding

P2X5 to be expressed on regenerating fibres and in particular on a sub-population of

activated satellite cells (myoblasts) (Ryten et al., 2004). Our laboratory also

demonstrated localized expression of specific P2X receptors in DMD and mdx

muscles with P2X1 and P2X6 co-expressed in small regenerating muscle fibres (Jiang

et al., 2005). Moreover, previous work in our lab has shown the expression of all P2X

mRNA transcripts at the 3 day, 4 week and 4 month stages of development in the mdx

mouse (Yeung et al., 2006), and a significant increase in expression of P2X7 protein

expression in 3 week mdx compared to normal (by quantification of

immunohistochemcial staining intensity) which were surmised to be in agreement

with previous reports of P2X7 expression in dystrophic macrophage lines (Ferrari et

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al., 1994) and their infiltration into degenerating dystrophic muscle (Kominami et al.,

1987). The perplexing observation had been the lack of a significant difference in

purinergic receptor protein levels, which could explain the significantly higher Ca2+

influx in response to high [ATP]e in mdx myoblasts compared with their wt

counterparts (Yeung et al., 2006). As described in Chapter 4, this, combined with

increased P2Y-type responses led to the hypothesis that the purinergic phenotype may

be due to lower ectoATPase activity and resulted in characterisation of the

extracellular hydrolysing potential of these cells (see Chapter 4). This analysis

however showed no significant difference that could explain the dystrophic purinergic

phenotype in mdx cells. It was only when quantitative RT-PCR and a novel C-

terminal rabbit polyclonal P2X7 antibody (Synaptic Systems) were used that a

significant up-regulation in P2X7 receptor expression was found in dystrophic

myoblasts compared with their C57 counterparts (see Chapters 5 and 6). The use of

immortalised myoblast cultures invites obvious questions as to the physiological

relevance of any conclusions drawn from such a model. Hence the immediate

requirement was to confirm that our observations could be extrapolated from our

immortalised cell cultures to primary myoblasts and to the organs from which these

were derived. Therefore, levels of P2X7 protein expression in C57 and mdx hind-

limb primary myoblasts and in muscle groups were analysed by Western blot using

freshly dissected tissues. 4 months of age was selected as the time point for this

analysis due to the absence of high grades of mononuclear (immune) cell infiltrations

in muscles at this age, contrary to younger mdx animals (Coulton et al., 1988,

Carnwath and Shotton, 1987). At 4 months, the majority of infiltrating cells (and

macrophages in particular) disappear from mdx muscles (Yeung et al., 2004). This

was imperative, as sub-populations of cells infiltrating dystrophic muscle have been

shown to express both P2X4 and P2X7 receptors (Yeung et al., 2004, Yeung et al.,

2006) which might influence our attempts to study P2X7 receptors in myogenic cells

themselves. Hind limb muscle groups were selected for analysis due to the large body

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of literature available describing the dystrophic phenotype in these muscles at varying

ages. Therefore, P2X7 protein expression was assessed in TA, GC, Sol and FDB

muscle groups, as well as in, diaphragm, as this muscle is known to show more

continuous degeneration throughout mdx life.

8.2 Results

8.2.1 Analysis of P2X7, ERK1/2, phospho-ERK1/2, and F4/80 protein

expression in normal and mdx muscle groups at 4 months

Abnormally high levels of P2X7 receptor expression have been so far show

here to be upregulated at the mRNA and protein level in immortalised and primary

myoblast cultures in vitro. This chapter serves to extend the PCR-based analysis of

P2X7 receptor expression in mdx muslces in situ described in Chapter 5 by examining

P2X7 receptor protein expression in tissues from the same animals. Western blot

analysis of 4 month C57BL10 and mdx- muscle groups showed P2X7 receptor protein

expression to be significantly up-regulated in four out of six muscle groups tested:

TA, GC, soleus and diaphragm, displayed fold changes in mdx vs.wild-type

expression of 2.4, 2.6, 2.2 and 2.5, respectively (Figure 8.2.1; A and C; *P<0.001).

Co-existent, significant elevations in basal ERK1/2 phosphorylation status were

observed in mdx TA and soleus muscle groups compared with those of age-matched

wild-type animals, with fold changes of 1.6 and 1.4, respectively (*P<0.01) (phospho-

ERK1/2 normalised to total ERK1/2; Figure 8.2.1; C). ERK1/2 levels did not

significantly differ between wild-type and mdx GC muscle groups (P>0.05; Figure

8.2.1; C), although this would appear to be due to higher experimental variability

rather than a tissue specific effect considering the SE variance involved. Significantly

elevated P2X7 receptor expression was also detected in mdx diaphragm muscles of

the same animals (Figure 8.2.1; B and C; *P>0.001).

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No significant differences were found in pan macrophage marker F4/80

protein expression between individual muscle groups of age matched normal and

dystrophic animals (P>0.05; Figure 8.2.1; E), and indeed, F4/80 expression proved

undetectable in all muscle groups using the immunoblotting technique.

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145

Figure 8.2.1. P2X7 expression and signalling in wild-type and mdx muscles at 4 months.

Representative immunoblots showing levels of dystrophin (Dp427), P2X7, phospho-ERK1/2

and ERK1/2, in specific muscle groups (TA, GC and soleus) of 4 month old mdx and

C57BL10 (WT) mice (A) and P2X7 expression levels in diaphragm from the same animals

(B) – Densitometric analysis of western blots analysed by ANOVA revealed significantly

higher levels of P2X7 receptor expression in TA, GC and soleus and diaphragm muscles and

basal ERK1/2 phosphorylation levels were also significantly higher in mdx TA and soleus

muscles compared with age matched controls (B and C, respectively;*P<0.001). The pan

macrophage marker F4/80 proved undetectable in immunoblots of protein extracts from the

mdx/C57 muscle tissues used in A+B (E). F4/80 immunostaining was however detected in

small localised regions of mononuclear infiltration in both C57 (F) and mdx (G) cryosectioned

TA muscle (representative images), visualised by colorimetric peroxidase staining (dark

pink/purple; F and G) with methyl green nuclear counter stain (blue). Primary antibody was

omitted for negative staining control (H).

8.3 Discussion

This study has reported for the first time that the P2X7 receptor protein

displays a pattern of significant and tissue-specific up-regulation in the dystrophic

mdx muscles at 4 months of age where, in general mdx muscles in situ clearly have

higher levels of P2X7 expression and show significantly enhanced ERK signalling,

which corresponds well with this increased P2X7 receptor expression levels found.

The possibility that infiltrating cells of the immune system (which, like

macrophages are known to express P2X7 receptor) might be responsible for the up-

regulations of P2X7 expression seen here in myoblasts and muscle in-situ has been

ruled out by probing the P2X7 immunoblots with anti-F4/80 antibody, a pan

macrophage marker (Khazen et al., 2005). These experiments showed no discernable

levels of F4/80 expression in any muscle groups tested compared with a very strong

specific signal detected in C57BL10 spleen (Figure 8.2.1), which is in agreement with

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other immunolocalisation data from our lab and from others. Indeed, at 4 months of

age, the hind limb muscle groups of the mdx mouse contain very low numbers of

infiltrating cells with few discernable areas of infiltration, compared with the earlier

periods (3-8weeks), when infiltrations of mononuclear cells are large and pronounced.

Figure 8.2.1; F-G shows that macrophages are indeed present in both C57 and mdx

TA muscle at 4 months, though limited to a small number of small localised areas.

Hence, infiltrating macrophages can be considered insufficient to explain the P2X7

expression in these muscles. The levels of P2X7 receptor expression in the

populations of macrophages that are present are however unknown, but for such large

up-regulations in P2X7 receptor expression to be explained by the possible existence

of such a small numbers of seemingly undetectable cells seems rather implausible. It

is even less likely in light of the demonstration of significant up-regulations in P2X7

receptor expression in immortalised and primary cultures of dystrophic myoblasts (see

Chapter 6), which would point to P2X7 elevation occurring directly in myoblasts and

myotubes. Importantly, P2X7 upregulation in dystrophic myotubes has recently been

confirmed in studies by Rawat et al, (2010).

The extrapolation of the previously discussed up-regulations in P2X7 receptor

expression found in mdx-derived myoblast cultures to mdx muscle in vivo suggests

that some significant physiological relevance could be linked to this phenomenon.

The P2X7-mediated component of the purinergic response in myoblasts has been

generally ignored until now; hence any predictions relating to functional

consequences of P2X7 receptor expression on these cells are completely novel. The

P2X7-mediated effects may potentially have been identified before, but mistakenly

attributed to P2X5 receptor-mediated responses (as discussed in Chapter 6).

Characterisation of the functional consequences of P2X7 receptor activation in muscle

in vivo are ongoing, however recent studies have demonstrated P2X7-mediated

inductions in myoblast proliferation and increased P2X7 mRNA transcript expression

levels upon differentiation in C2C12 cells (Martinello et al., 2011). These responses

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are clearly multifactorial as pharmacological P2X7 receptor inhibition in vitro has

been shown to inhibit myotube formation in C2C12 cells, but only when cultured in

differentiation inducing low serum media (Araya et al., 2004). Therefore, the up-

regulated P2X7 receptor expression levels described here in dystrophic myoblasts and

muscles in situ may have a significant role to play in the pathology of dystrophic

muscle, potentially dictating the proliferation/differentiation status of muscle resident

stem cell populations. However, although the heightened sensitivity of dystrophic

myoblasts appears to be an inherent attribute of these cells, the factors orchestrating

cascade activation may be intricately complex and dictated by potentially quite

localised changes in extracellular milieu. Whatever the functional consequence of the

descried phenomenon, the results presented here reinforce the notion that the elevated

levels of P2X7 expression detected in mdx muscles are indeed of muscle-specific

origin and not an artefact of small numbers of infiltrating cells.

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9

Analysis of the effects of pharmacological

blockade of P2X7 receptor in mdx mice in vivo

9.1 Introduction

ATP, present in cytosol at millimolar concentrations, was first examined as a

potential mediator of pathological effects by (Burnstock, 1996) in relation to its role

as a chemical mediator of nociception. The same group considered the potential

consequences of release of millimoles of ATP from the dystrophic muscle, proposing

roles for P2X5 and P2Y1 receptors in activated satellite cells during muscle fibre

regeneration (Ryten et al., 2004). Our group has extended this line of investigation

with the findings that P2X7 receptor expression and activity are significantly

increased in both myoblasts and adult tissues derived from the mdx mouse model of

DMD, suggesting a possible role for this receptor in the dystrophic pathology.

Apoptosis has long been speculated to potentiate the dystrophic pathology in skeletal

muscle, largely associated with increases in intracellular calcium (Matsuda et al.,

1995, Tidball et al., 1995, Spencer et al., 1997). As previously discussed, P2X7

receptors have well documented roles in the initiation of apoptosis (Di Virgilio et al.,

1996) for a review, hence the possibility exists of similar pathways operating in

skeletal muscle, where ATP concentrations released into extracellular space would be

high and thus the effects of purinergic signalling even more significant. However,

P2X7 receptors have also been shown to confer growth advantages in different

circumstances or specific cell lines (see Di Virgilio et al., (2009) for a review),

leading to the current hypothesis that P2X7 receptors may have a bi-functionality;

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conferring metabolic and proliferative advantages during sustained low level or tonic

stimulation and changing into the initiation of apoptotic cascades in response to short

bursts of high level stimulation. It is possible that both mechanisms may be operating

in dystrophic muscles.

The discovery of an up-regulation in functional P2X7 receptor expression in

dystrophic muscle indicates a possibility of therapeutic intervention through the

pharmacological blockade of P2X7 signalling in vivo. CBBG is a commonly used

and highly selective antagonist of the P2X7 receptor (Jiang et al., 2000),

demonstrating low in vivo toxicity over sustained periods (Remy et al., 2008, Taylor

and Thorp, 1959), and as such, was a good candidate for blocking P2X7 activation in

dystrophic muscle. The parent compound, CBB, has been shown to be non-toxic in

the mouse over extended periods when injected intraperitonealy (i.p.) up to 500mg/kg

(Taylor and Thorp, 1959). More recently, specific in-vivo P2X7 receptor blockade

using CBBG injected IP in rats at concentrations of 50mg/kg (Peng et al., 2009) and

100mg/kg (Gourine et al., 2005) has shown beneficial effects under pathological

conditions of high concentrations of extracellular ATP release into the extracellular

environment in damaged or necrotic spinal cord and systemic inflammation.

However, due to the lack of an in-vivo study of the half-life or metabolism of this

compound in mice, an initial LCMS analysis of CBBG levels in the plasma of

injected animals has been performed (data not shown). Since most of the CBBG in

plasma was found bound to proteins and due to species-specific differences in

receptor sensitivities, a concentration of 125mg/kg body weight was adopted for this

study. Two injection regimens were employed to study the short and long-term effects

of receptor blockade short term (daily for 30 days, starting at 3 weeks of age), or long

term (weeks 3-14, every third day). In both cases injections of CBBG or saline

solution were given i.p., with subsequent histological analysis of the effect of in-vivo

P2X7 receptor blockade on both the percentage central nucleation, as a marker of the

previous occurrence of necrosis (Karpati et al., 1989), and also the percentage of

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revertant fibres as a marker of myogenic precursor activity and previous

pathologically-induced regenerative activity (Yokota et al., 2006, Hoffman et al.,

1990, Wilton et al., 1997).

9.2 Results

9.2.1. Analysis of relative proportions of revertant and centrally nucleated

fibres in mdx TA muscle following in vivo CBBG injection

In order to asses the efficacy of pharmacological P2X7 inhibition in vivo, the

well established methodology of enumerating centrally nucleated and revertant fibre

numbers was applied to cryosectioned TA muscle sections immunostained with anti-

dystrophin antibody (DSHB). Manual counts of central nucleation, revertant fibre

number and total fibre number were obtained using „ImageJ, cell counter plugin‟

(Rasband, 1997-2005). Percentage counts were obtained for numbers of revertant and

centrally nucleated fibres from total fibre counts per transverse muscle section and

averages obtained from multiple sections taken from similar areas of each muscle.

This data was analysed using two-way ANOVA with post-hoc Tukey‟s test (Origin

7.0). In the long-term injection group ANOVA revealed no significant differences in

average percentage central nucleation of CBBG compared with saline injected mdx

muscles of either age group (P>0.05), although an expected significant increase in

percentage central nucleation was observed in older animals compared with younger

ones (Figure 9.2.1; top left panel; *P<0.0001). No significant differences were found

in revertant fibre numbers between CBBG and saline-injected muscles at 6 weeks of

age (P>0.05), but 2 way ANOVA revealed a significant decrease in revertant fibre

number recorded in CBBG-injected compared with saline injected muscles at 16

weeks (Figure 9.2.1; top right panel; *P=0.028 and 0.0003 for treatment and age,

respectively).

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Figure 9.2.1. Pharmacological inhibition of P2X7 receptors in vivo - effect on mdx

muscle morphology. Graphical representations depicting the effect of in vivo CBBG

administration on mdx TA muscle. ANOVA revealed that differences in central nucleation

did not differ significantly between CBBG and saline (control) injected animals (P>0.05),

although a significantly higher percentage central nucleation was observed in 16 week old

animals compared with those of 4 weeks of age (upper left panel; *P<0.0001). CBBG had no

significant effect on the number of revertant fibres observed in 4 week old animals (P>0,05),

yet a significantly higher percentage of revertant fires was observed in CBBG injected 16

week old animals compared with age matched saline injected controls (upper centre panel;

*P<0.0001). No significant differences were found in average revertant fibre number per

revertant colony (upper right panel; P>0.05). Lower panel micrographs illustrate central

nucleation (lower left panel) and revertant fibre identification by dystrophin

immunohistochemical staining (lower right panel).

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9.3 Discussion

The finding that percentage revertant number is significantly lower in CBBG-

injected 16 week old mdx mice compared with saline injected age matched controls

could suggest that the inhibition of P2X7 mediated signalling effected a decline in

proliferative potential of myogenic precursors in this muscle, indicating that P2X7

receptors may indeed provide a potential growth advantage in mdx muscle of this age.

Alternatively, it could suggest that muscle fibres of CBBG injected animals from this

group may have been subjected to fewer cycles of degeneration/regeneration than the

saline injected controls due to a reduction in the P2X7-mediated arm of the

apoptotic/necrotic process which may be active during the earlier

degenerative/regenerative phases in these muscles. Moreover, the impact of treatment

on myofibre- and macrophage-mediated responses cannot be distinguished here, with

both mechanisms co-existing in dystrophic muscle. The reduction in revertant fibre

number was restricted to the group of 16 week old animals, injected over a longer

period, and not significant in the 4 week old group, which could suggest that the

effects of inhibition may only be manifested slowly, as would be expected due to the

very low levels of revertant fibres normally present in adult mdx muscle: 1-5% as

derived from this study, which is in agreement with figures quoted by other

investigators (<5%; Dr Susan Brown, personal communication). Hence, the small yet

significant decrease in revertant fibre expression by ~1% in the CBBG injected 16

week old mdx muscle may be indicative of a much larger effect on total proliferative

activity of the myogenic precursor cells in these muscles, or indeed of the extent of

P2X7 receptor‟s role in proliferation/differentiation responses. Another possibility

may be that non-P2X7 mediated effects may have masked the specifically myogenic

effect of CBBG inhibition. At 4 weeks of age these muscles would contain high

levels of infiltrating mononuclear cells. Macrophage-mediated effects in dystrophic

muscle have been widely studied, but, paradoxically, not all have been reported to be

detrimental; 4 week mdx muscle has been reported to contain M1 macrophages

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responsible for lysing myoblasts via NO-mediated mechanism, as well as M2

macrophages that reduce lysis through competition for arginase (Villalta et al., 2009).

The P2X7 receptor expression levels in these two populations has not been assessed

and it is not clear which population has the dominant effect in-vivo, so it remains

impossible to predict the effect of CBBG-mediated P2X7 receptor blockade in-vivo

on either population, or the macrophage-mediated component of the

degenerative/regenerative process in general.

The significant increase in percentage central nucleation with increasing age

was in agreement with the age related pattern of necrosis described by Karpati et al.,

(1989), which was shown to peak at around 6 weeks of age. Thus, the group of 16

week old animals would have more time in which regeneration could occur, hence the

cumulative phenomena of central nucleation observed (Yokota et al., 2006). No

significant differences in percentage central nucleation were observed between CBBG

and saline injected muscles in either age group, suggesting that the mechanism by

which satellite cells become and remain activated and centrally nucleated may differ

from that by which they proliferate and therefore undergo somatic mutations that give

rise to revertant fibres – this is due to the fact that a significant decrease in revertant

fire number was recorded in CBBG injected mdx muscle from those animals receiving

the longer injection course. This could suggest that the myofibres of CBBG injected

animals had undergone fewer degenerative/regenerative cycles and therefore

displayed a reduction in revertant fibre number as a result of this (Yokota et al.,

2006). Alternatively, it could also potentially mean that CBBG had an anti-

proliferative effect on the satellite cell population of dystrophic muscle, without

affecting their potential for becoming centrally nucleated. This would indeed be in

agreement with the observation that myostatin is able to increase myofibre size

without increasing satellite cell number (Amthor et al., 2009). Hence CBBG may

have had a influence over the proliferative but not the differentiation potential of

dystrophic satellite cells, a phenomenon recently observed through the

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pharmacological inhibition of P2X7 receptor signalling in vitro (Martinello et al.,

2011). The demonstration by this study that a reduction in revertant fibre number in

CBBG injected animals is not accompanied by any change in average revertant fibre

number per revertant colony would be in agreement with the idea that proliferation

and migration/differentiation are separately regulated in muscle stem cells, however,

the extent to which the factors of proliferation and differentiation affect revertant fibre

colony size has not been explored in the literature to date.

Support for the notion of ATP-mediated pathology progression, and

pharmacological inhibition of P2X7 receptor signalling conferring beneficial effects

in dystrophic muscle in vivo, can also be found in the demonstration that suramin

injection (a less specific P2 receptor antagonist) reduces both muscle damage and

fibrosis in the mdx mouse (Taniguti et al., 2011, Iwata et al., 2007). One final

noteworthy point is that visual inspection following dissection consistently

demonstrated an almost complete lack of evidence that CBBG was able to effectively

penetrate the muscles of injected animals. All other major organs, peritoneal cavity,

and even skin appeared very distinctly blue, whereas the skeletal muscles of the hind

limbs remained virtually normal in appearance. This would suggest that rather than

insufficient dosage, the low level of observable effect is more likely due to the

inefficient permeation of the dye into skeletal muscle tissue. The obvious way

forward in this instance would be to try intra-muscular injections. However, no

obvious significant differences in pathology were observed upon preliminary

histological examinations of diaphragm muscles from CBBG injected mice, where the

degree of dye penetration following i.p. injection would be assumed to be higher.

Moreover, the characterised expression of multiple functionally active P2X7 splice

variants in mdx TA muscle may suggest that the use of any P2X7 inhibitor in vivo is

belayed by the requirement for elucidation of the splice variant selectivity of such

drugs.

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10

General Discussion

10.1 Introduction

Mutations in the DMD gene encoding dystrophin, are responsible for the most

common congenital muscular dystrophy (O'Brien and Kunkel, 2001) and are

sufficient to confer often co-existing deficiencies in muscular ambulation, respiration

and circulation as well as central nervous system defects leading to variable degrees

of specific cognitive and vision impairment (Anderson et al., 2002, Emery and

Walton, 1967, Fitzgerald et al., 1994, Costa et al., 2007). The dystrophin protein

itself classically represents the primary point of anchoring for a large complex of

proteins known as the dystrophin associated protein complex (DAPC) at the muscle

sarcolemma. Mutations and subsequent functional loss of this protein generates a

compound phenotype through loss of DAPC complex interactions, with a range of

specific mutations generating phenotypic diversity (Ohlendieck et al., 1993,

Ohlendieck et al., 1991). Loss of function mutations in the dystrophin gene are

responsible for the commonest and severest of the muscular dystrophies in humans

known as Duchenne Muscular Dystrophy (DMD) (Monaco et al., 1986, Hoffman et

al., 1987). Less frequent in-frame mutations of the same gene result in partially

functional truncated dystrophin protein products responsible for the milder Becker‟s

Muscular Dystrophy (BMD) (Hoffman and Kunkel, 1989). Precise elucidation of the

specific molecular mechanisms underlying any given disorder is paramount to

therapeutic development, and further insights into the cellular and molecular

abnormalities in DMD pathology are essential to understanding the causative

mechanistic events surrounding the phenotypic abnormalities of dystrophic muscle.

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10.2 Role of ectoATPases in DMD

LGMD2D - attributed to mutations in the protein product of the -SG gene,

presents a similar phenotype to that of DMD, though occurring without loss of the

dystrophin-glycoprotein complex-F-actin link (Roberds et al., 1994). Since -SG has

been ascribed one known function, that of an ecto-ATPase enzyme (Sandona et al.,

2004, Betto et al., 1999) it thus provides a potential insight into the extracellular

nucleotide-mediated component of DMD pathology without the compounding factors

arising from membrane instabilities due to dystrophin complex loss; Importantly, this

ecto-ATPase activity is particularly pronounced at high levels of extracellular ATP

[ATP]e leading to the hypothesis that abnormalities in purinergic signalling may

operate in dystrophic muscle, through the reduction of extracellular nucleotide

metabolism following loss of –SG from the sarcolemma. Such a phenomenon

would occur in both the absence of dystrophin and directly from -SG mutations.

Our laboratory has previously identified a heightened sensitivity to ATPe in an

immortalised dystrophic myoblast cell line, which was shown to be P2X receptor

dependent – mediated through influxes of extracellular Ca2+

. Yet a P2Y component

was also present and despite the documented functional abnormality of a specific

receptor subtype, no differences in receptor expression patterns had been found in

normal and dystrophic myoblasts (Yeung et al., 2006). Following vesicular or

hemichannel-mediated release, the availability of ATP and other nucleotides is

modulated through their rapid degradation by the ectonucleotidase family of

membrane proteins (Burnstock, 2009) leading to the hypothesis that abnormalities in

the routine hydrolysis mechanisms, which normally tightly regulate the levels of all

extracellular nucleotides, may confer localised nucleotide over-sensitisation sufficient

to explain the altered responses documented in dystrophic myoblasts. However,

results of this study excluded the effect of differential extracellular nucleotide

hydrolysing potential as explaining the heightened purinergic sensitivity of dystrophic

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myoblasts. Instead, this work serves to highlight the novel role of specific P2X7

receptor expression abnormalities in conferring heightened ATPe sensitivity to

dystrophic myoblast cells – where significant up-regulation in P2X7 receptor

transcript expression and coexistent up-regulation in protein expression have been

documented in mdx-derived primary myoblasts, immortalised myoblast lines and

muscles in situ.

10.3 Role of P2X7 receptors in DMD

Although not unequivocally causative of any particular pathology, P2X7

receptors have been implicated in multiple pathological disorders (Khakh and

Burnstock, 2009) and possess a diverse array of functions; coordinating fast excitatory

transmission in the central and peripheral nervous systems, orchestrating controlled

changes in circulation via smooth muscle/endothelial-mediated contraction, and

perhaps principally in the induction of cellular responses in immune cells such as

lymphocytes, macrophages and microglia (Burnstock, 2007, Burnstock, 2009). P2X7

receptor expression has been shown to be widespread in tissues and cells of the

immune system where they regulate the processing and release of cytokines and

inflammatory mediators (Di Virgilio, 2007, Di Virgilio et al., 1996), and their

uniquely lower sensitivity to ATP has made them the focus of intense interest in

arenas of pathological inflammation, tissue degradation and cytolysis. Unlike other

P2X receptors, P2X7 responds only when high ATP levels are present. To that end

P2X7 has become synonymously a „danger‟ sensor, with well documented roles in the

development and potentiation of the immune system‟s inflammatory response. The

cellular/functional consequences of P2X7 activation have been widely studied in

lymphocytes and macrophages and include: morphological changes via actin filament

rearrangement (Pfeiffer et al., 2004), membrane blebbing (Wilson et al., 2002),

cytoloysis/apoptosis (Di Virgilio, 2000), IL-1 processing and release (Ferrari et al.,

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1997), lysosomal impairment and autophagolysosome release (Takenouchi et al.,

2009) and the conferment of proliferative advantages through mitochondrial

depolarisation (Di Virgilio et al., 2009). Specifically in terms of myofibre

regeneration, an important feature of P2X7 receptor function may be the dual nature

of P2X7 activation, where a low level, tonic ATP stimulation is conferring metabolic

and proliferative cellular advantages, whereas elevated to high levels and/or sustained

or chronic stimulation is able to trigger cytolysis, apoptosis or autophagic cell death.

Two mediators of such a functional balance have been suggested: [Ca2+

]i mobilisation

and opening of a large P2X7-dependent membrane pore through some still

uncharacterised mechanism(s) (Di Virgilio et al., 2009).

It is now widely recognised that ATP is not only released by dead or dying cells but

also physiologically, by live cells including muscle (Becq, 2010). Indeed, high

concentrations of ATP released during muscle contraction have been shown to

modulate Ca2+

homeostasis and muscle physiology, acting through purinergic

receptors (Buvinic et al., 2009). Several purinergic receptors have been found

differentially expressed in myoblasts, myotubes and muscle fibres at different stages

of development (see below). More recently, P2X7 has emerged as physiologically

important skeletal muscle receptor. Its activation stimulates myoblast proliferation

(Martinello 2011) while its inhibition in differentiating cells prevented myotube

formation (Araya et al., 2004). As mentioned earlier, changes in functional P2X7

receptor expression have been documented in various pathological environments (Di

Virgilio, 2007, Di Virgilio et al., 2009), also including the muscle of the dysferlin

deficient mouse model of LGMD2B (SJL/J mouse). In this mouse up-regulated P2X7

receptor expression was linked to LPS/BzATP-induced IL-1 secretion from

myoblasts, and inflammasome formation (Rawat et al., 2010). In the same study,

Rawat‟s group also documented the up-regulated expression of P2X7 receptor protein

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in cultured mdx myotubes, (surprisingly, mdx myoblasts were not examined although

SJL/J myoblasts were analysed). The present study extends our understanding of the

roles for ATPe in DMD disease pathology, specifically in the mdx mouse model,

where up-regulation of P2X7 receptor expression in dystrophic myoblasts has been

shown to confer heightened functional responses. These responses could explain

several of the observed abnormalities in dystrophic muscles.

The well documented roles of P2X7 receptors in ATP-induced lytic cell death and

in immune-modulators release are of particular relevance in muscle, where ATP and

indeed NAD are essential facilitators of muscle metabolism. Even more so in

dystrophic muscle, where degenerating fibres provide potential sources of

extracellular ATP at concentrations exceeding that which could be achieved anywhere

else in the body. Differing outcomes of P2X7 receptor responses are thought to result

from low level or tonic stimulation, compared with sudden or prolonged high level or

excitotoxic ATPe stimulation (Di Virgilio et al., 2009). The heightened P2X7

receptor responses in dystrophic myoblasts may therefore potentially influence the

balance between positive and negative ATP-induced effects on the stem cell pool of

dystrophic muscle; where induction of proliferation, differentiation and apoptosis

could result (Martinello et al., 2011, Araya et al., 2004). Indeed, tonic P2X7 receptor

stimulation has been shown to induce a slow, controlled influx of Ca2+

, promoting

healthy metabolism and growth while avoiding the catastrophic effects of un-checked

Ca2+

entry. Specifically, beneficial effects have been proposed to include the Ca2+

-

induced enhancement of mitochondrial energy production, ER Ca2+

accumulation and

increased cytosolic [ATP]i, promoting serum independent growth following basal

autocrine P2X7 receptor stimulation (Di Virgilio et al., 2009, Adinolfi et al., 2005);

an effect proposed to occur via a calcineurin regulated induction of the „nuclear factor

of activated T cells‟ (NFATc1) nuclear translocation in muscle (Adinolfi et al., 2009,

Sakuma et al., 2003). Calcineurin was identified in the mass spectrometry analysis

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conducted for this thesis as a protein showing significantly increased levels of

phosphorylation in ATP treated dystrophic myoblasts. Calcineurin phosphorylation

(and subsequent activation) has previously been demonstrated in an ERK dependent

manner (Aramburu et al., 2004), suggesting a potential role for P2X7 in calcineurin-

NFAT mediated hypertrophy in dystrophic myoblasts. Indeed, it has been suggested

that the limited beneficial effects of glucocorticoid treatment in DMD are due to the

activation of the calcineurin-NFAT pathway (St-Pierre et al., 2004). Interestingly,

one of the issues raised by Di Virgilio‟s group, who focussed primarily on transfected

HEK-293 cells, was the unknown origins of the low [ATP]e which would be able to

explain the existence of such autocrine mechanisms in vivo – yet active/passive ATP

release from muscle cells is well documented (Becq, 2010, Buvinic et al., 2009) and

would indeed be pronounced in dystrophic muscle due to contraction-induced ATP

release via both secretory and necrotic pathways (Allen et al., 2010b, Allen et al.,

2010a, Allen et al., 2005, Head, 2010).

ATP has long been known to be released by skeletal muscle both dependently and

independently of contraction (Forrester and Lind, 1969) with specific release later

demonstrated from neuromuscular junctions (Silinsky, 1975) and more recently, in

cultured myoblasts and myotubes (Martinello et al., 2011). Although the potential

existence of extracellular ATP receptors in skeletal muscle was first demonstrated

over a quarter of a century ago (Kolb and Wakelam, 1983), only now are its

intricacies beginning to be resolved, highlighting the depth of signalling complexity to

be explored. The proposal that extracellular ATP may be an important mediator of

satellite cell plasticity stems from the regulated changes in expression of P2X5 and

P2X6 receptors in developing chick muscle (Meyer et al., 1999) and P2X2, P2X5 and

P2X6 receptors in developing rat muscle (Ryten et al., 2001). The same group

proposed the P2X5 mediated induction of differentiation in C2C12 cells (Ryten et al.,

2002), and implicated P2Y1, P2Y4, P2X2 and P2X5 receptors in the regeneration of

adult skeletal muscle, illustrating the sequential expression of P2X5, P2Y2 and P2X2

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receptors in regenerating mdx muscle in situ. However, somewhat surprisingly, the

possibility of macrophage infiltrations i.e. cells expressing these receptors, in 5 week

mdx muscle was never addressed (Ryten et al., 2004). P2X2 and P2Y1 and P2Y2

receptors have been implicated in neuromuscular junction formation, stabilisation and

function (Choi et al., 2003, Voss, 2009, Ryten et al., 2007) and the expression of all

P2X receptor subtypes has been documented in cultured myoblasts (Yeung et al.,

2006), although specific roles for other sub-types are unresolved at this time.

The functional involvement of P2X receptors in the differentiation of C2C12 cells

has been demonstrated via autocrine ATPe response – an effect that could be

replicated by oxidised ATP (oATP) application; a non-specific P2X inhibitor (Araya

et al., 2004). Moreover, it has recently been proposed that concentrations of ATPe are

significantly higher in the extracellular environments of myoblasts in comparison to

myotubes, as a consequence of a low ecto-ATPase activity in myoblasts (Martinello

et al., 2011). This minimal ATP hydrolzising activity of myoblasts was also found

here, illustrating the potential significance of up-regulated P2X7 receptor levels in

dystrophic myoblasts as shown here, especially in proximity to damaged/degenerating

myofibres, where the functional effects of such up-regulation would be accentuated

by elevated levels of [ATP]e. The observed effects of ATP in C2C12 cells do

however, appear to be multifactorial, with ATPe displaying opposing functions in

proliferating/differentiating cells. Martinello‟s group have demonstrated the P2X7-

dependent induction of proliferation in ATP/BzATP treated C2C12 myoblasts 24h

post serum removal. This effect was reversed by P2X7 receptor blocker oATP.

Araya‟s group, also using oATP, demonstrated the inhibition of differentiation in

C2C12 cells 4 days in differentiation medium, proposed but not confirmed to also be

a P2X7-dependent effect (Martinello et al., 2011, Araya et al., 2004). The reason for

these differentiation stage -dependent differences in P2X7 receptor function in C2C12

cells is unclear; no significant changes in P2X7 receptor expression upon

differentiation were documented by either group; Martinello showed P2X4 mRNA

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levels to be up-regulated upon differentiation, which could potentially explain the

induction of differentiation in C2C12 cells exposed to ATPe documented by Araya‟s

group, since oATP was the only antagonist used to inhibit the differentiation effect,

and oATP also inhibits many other P2X and P2Y receptors (Friedle et al., 2010).

Unfortunately, this group did not investigate the P2X4-mediated component of the

proliferation response in these cells, which could easily have been achieved with

ivermectin pre-treatment. Also interesting here is the fact that Martinello‟s group

showed that higher concentrations of ATPe proved toxic to myoblasts, which would

be in agreement with P2X7 receptor stimulation switching from tonically beneficial

to excitotoxically catastrophic effects (Di Virgilio et al., 2009). Martinello also

confirmed the importance of autocrine ATPe signalling in myoblasts, showing a

reduction in myoblast survival following apyrase treatment (triggering ATP

degradation). Together, these observations confirm the significance of the findings of

this study; that up-regulation in functional P2X7 receptor expression confers

heightened nucleotide sensitivity to dystrophic myoblasts, with the potential for

localised and differentiation-stage dependent induction of proliferation,

fusion/differentiation and cell death. Whether such responses in DMD are singularly

controlled by autocrine/paracrine ATPe signalling seems doubtful considering the

high levels of ATP released from degenerating myofibres, although the exact levels of

ATP release from normal and dystrophic myoblasts have not been addressed.

Studies are ongoing in our laboratory to determine whether the P2X7-dependent

stimulation of myoblasts falls into the proposed tonic versus excitotoxic mode. The

role of individual P2X7 splice-variants, specific subunit interactions, or indeed, novel

interacting signalling partners in such effects also remains to be seen - such has been

proposed in relation to pannexin-1‟s role in lytic pore formation (Shen et al., 2010),

an effect found here to be absent in proliferating myoblasts and in differentiated

myotubes, despite the expression of pannexin-1 (Figure Appendix 4).

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The potential for P2X7 receptors contributing to the dystrophic pathology was

assessed in this study via in vivo injection of the specific pharmacological inhibitor

CBBG. The significant reduction in revertant fiber number in mdx TA muscle

following CBBG injection could indicate that the number of

degeneration/regeneration cycles in these animals was reduced (Winnard et al., 1993,

Klein et al., 1992, Yokota et al., 2006). It could however, also indicate a role for the

P2X7 receptor in myoblast proliferation, as has been described above (Martinello et

al., 2011), thus reducing the regenerative potential of injected mdx muscles. This is

being investigated in vitro by pre-incubation of primary myoblast cultures with

CBBG prior to ATPe stimulation.

10.4 P2X7 splice variant expression and function in mdx muscle

P2X7k is the only mouse P2X7 splice variant to be characterised in the

literature - displaying a propensity for heightened agonist sensitivity and prolonged

activation leading to constitutive large pore formation and cell death in transfected

HEK-293 cells. Up-regulation of P2X7k variant expression has been documented

here in dystrophic myoblasts and muscles in situ, however, the true functional

relevance of P2X7k in vivo has never been investigated. Neither indeed has its

antagonist selectivity been documented, which, as alluded to in the chapter-specific

discussion, is surprising considering it‟s potentially significant contribution to cellular

responses in vivo (Nicke et al., 2009). No functional N-terminal P2X7 splice variants

have been characterised in humans to date – therefore no comparisons can be made.

Of the functional human C-terminal variants that have been described, P2X7B and

P2X7J have both been reported to confer proliferative gains, yet by distinctly

opposing mechanisms of function: P2X7B acts in the potentiation of P2X7A agonist

responses, promoting growth and proliferation (Adinolfi et al., 2010), and P2X7J in

the hetero-oligomeric inhibition of P2X7A receptor channel formation. This oddly

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enough has been suggested to confer similar gains of function in terms of the growth

and proliferation of tumour cells by inhibiting the apoptotic cascade induction of the

wild-type P2X7A response (Feng et al., 2006).In fact, a patent has been filed relating

to the application a monoclonal antibody raised against P2X7J in the treatment of

P2X7J- positive tumours. Though capable of forming functional ion channels, both

P2X7B and P2X7J are deficient in large pore formation. Their ability to hetero-

oligomerise with P2X7A could form the basis of a possible explanation for the

absence of large pore formation in the myoblasts described here. Indeed, if this is

further extended to the pattern of splice variant expression observed in mdx

diaphragm shown here - where mouse P2X7a was the only splice variant found to be

up-regulated, compared with limb muscles where up-regulation of P2X7a, P2X7b and

P2X7c was found to coexist. The possibility then exists for P2X7 splice variants to

convey significant functional differences for diaphragm muscle and its myogenic

progenitors. Although mdx limb muscle degeneration is progressive and associated

with a decline in replicative potential (Mouisel et al., 2010), mdx limb muscles do not

display the progressive fibrosis associated with the human pathology and mdx

diaphragm is widely regarded to better represent the human condition (Stedman et al.,

1991, Andreetta et al., 2006, Coirault et al., 2003). One could speculate that the

increased severity of disease progression in mdx diaphragm could be related to the

P2X7 splice variant profile observed in this muscle type: The lack of any potentially

dominant negative effect of P2X7b or P2X7c variant expression on the activity of

P2X7a receptors would leave unchecked the P2X7-triggered apoptotic cascade (Di

Virgilio, 2000), which, has previously been implicated in dystrophic satellite cell

death through an undefined mechanism (Merrick et al., 2009, Jejurikar and Kuzon,

2003). However, the validity of such functional extrapolations between human and

mouse variants remains to be confirmed. Conversely, the up-regulation of P2X7k

variant expression shown in this study could be involved in the mechanism by which

fibrosis progressively and preferentially accumulates in the mdx diaphragm but not in

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mdx limb muscles, which has previously been linked to the constitutive over-

proliferation of connective tissues (Coirault et al., 2003, Andreetta et al., 2006).

10.5 P2X7 expression in myogenic precursors

Satellite cells comprise approximately 2-7% of the nuclei associated with a

particular myofibre in an adult muscle (Peault et al., 2007). The proposal of Pax7

transcription factor expression being a unique requirement for adult satellite cell

function originated in the observation that Pax7 knockout mice showed no significant

abnormalities in foetal myogenesis, whereas their postnatal muscle growth was

severely impaired (Seale et al., 2000). As such, Pax7 has become a seminal marker

for adult satellite cell populations in vivo (Oustanina et al., 2004, Peault et al., 2007,

Zammit et al., 2006), although in vivo confirmation of its function in adult muscles

have been questioned by others (Lepper et al., 2009). More recently, other

populations of myogenic precursor cells have been shown to inhabit distinct niches in

adult skeletal muscle outside the normal satellite cell-associated region beneath the

basal lamina. Thus, pericytes have been shown to possess the capacity to transverse

from their normal endothelial locations within blood vessels, crossing the

extracellular matrix to reach mature myofibres. They are able to participate in

myofibre regeneration through the expression of myogenic regulatory factors such as

Myf-5 and by concomitant fusion to form new myofibres (Dellavalle et al., 2007).

PW1+/Pax7

- interstitial cells (PICs) are the latest in this line of novel myogenic

progenitor cell populations to be proposed. Residing solely in the interstitial space

between mature myofibres and characterised by a lack of Pax7 expression, PICs are a

muscle-resident stem cell population distinct from satellite lineage and able to

participate in the fusion and regeneration of mature skeletal muscle (Mitchell et al.,

2010). Without completely ignoring the rather attractive, yet unfounded notion that

the P2X7+/ Pax7

- cell population that we have described could represent anther novel

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population of myogenic progenitors at similarly distinct location, perhaps a more

logical hypothesis may be that the cells defined here could indeed be a population of

activated PICs taking residence externally to the basement membrane in response to

some internal stimulus. They are unlikely to be pericytes since they appear to express

Myf-5 (Dellavalle et al., 2007). Transcription factor expression profiling of P2X7

receptor expressing cells on a single isolated living fibre and derived myoblast

primary cultures may shed some light on whether the cells identified in this study

represent a homogeneous population of myogenic precursor. PCR using laser

dissected mononuclear cells attached to living myofibres ex vivo could also be used

here. Perhaps a more elegant study could employ the single cell based oil-water

emulsion PCR methodology gaining popularity in the high throughput analysis of

complex microbial and cancer cell specialisations (Zeng et al., 2010, Novak et al.,

2010).

10.6 Consequences of altered P2X7 expression and function in dystrophic

muscle

Abnormalities in ERK signalling cascade described here in relation to

dystrophic myoblasts are likely to have consequences for a host of cellular responses.

These could be broadly categorised by bifold signalling cascade induction leading to

increased survival/growth or apoptosis/autophagy/necrosis and cell death (Mebratu

and Tesfaigzi, 2009). It has been shown that MAPK family members are responsible

for determining the activation state of satellite cells in adult muscle, with quiescence

suggested to involve the p38/ phosphatase MKP-1 (Jones et al., 2005). Ablation of

this enzyme has been shown to exacerbate the dystrophic phenotype in mdx muscle

(Shi et al., 2010). However, the dependence of MKP-1 on P2X7 activation remains to

be seen. The multi-faceted nature of MAPK signal cascade in satellite cells is

illustrated by the fact that ERK1/2 activation induces both proliferation and

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differentiation of cultured myoblasts (Gredinger et al., 1998, Jones et al., 2001,

Milasincic et al., 1996). Yet these observations have been proposed to be cell type

specific: ERK1/2 activation induces proliferation in C3H-derived myoblasts, and

differentiation in 23A2-, L6A1- and C2C12-derived myoblasts (Jones et al., 2005).

The demonstration of P2X7-dependent ERK1/2 activation in myoblasts in this study,

combined with the more recent demonstration that ATPe can induce either

proliferation or differentiation of C2C12 myoblasts under different conditions via a

mechanism proposed to be P2X7-dependent (Araya et al., 2004, Martinello et al.,

2011) suggest that the previously observed differences in MAPK-mediated response

in myoblasts may indeed be due to differential expression/activation of P2X7 receptor

levels/variants between different cell lines or differences in conditions of individual

cultures.

Besides heightened P2X7 receptor sensitivity, this study has also highlighted the role

of NAD-mediated P2X7 receptor activation in dystrophic myoblasts. NAD has

previously been shown to induce ATP-independent Ca2+

influx via ADP-ribosylation

of ATP binding sites of P2X7 receptors (Domenighini and Rappuoli, 1996). This

mechanism is active in murine T cells, yet in macrophages, NAD does not serve to

gate the channel, it only decreases the gating threshold in response to ATP binding,

demonstrating synergistic interaction between ATP and NAD to induce P2X7

signalling cascades (Seman et al., 2003b). NAD-mediated cell death has been

demonstrated in a variety of disorders (Divirgilio et al., 1989, Zhao et al., 2011,

Seman et al., 2003b). Here dystrophic myoblasts have been shown to display

heightened NAD sensitivity, the functional relevance of which is under investigation.

As already covered in the chapter-specific discussion, ART1 is thought to be the

predominant ADP-ribosyl transferase expressed in skeletal muscle, where its

expression has been shown to be up-regulated upon differentiation of C2C12 and

C3H-derived myoblast cultures (Zolkiewska et al., 1992, Friedrich et al., 2008).

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Experiments are underway to determine ART1 expression levels in mdx muscle and

derived primary myoblast cultures.

In an attempt to broaden the current knowledge and elucidate the immediate

functional consequences downstream of P2X7 receptor activation in myoblasts, a

mass spectrometry based approach was employed here in assessing protein

phosphorylation states between normal and dystrophic myoblasts following ATP

treatment. Several proteins were identified as novel participants in the ATPe response

in dystrophic muscle, notably the ERM actin binding proteins, annexin A6 and PKC.

The ERM proteins have well documented roles in cell shape changes, motility and

proliferation (Bretscher et al., 2002). They have received recent attention because of

their potential roles in cancer biology due to similarities with another ERM family

member, the tumour suppressor protein, merlin (McClatchey, 2003, Morales et al.,

2010, Ren, 2010), although there is no current literature linking P2X7 receptors and

ERM proteins directly.

P2X7-dependent PKC phosphorylation has previously been reported to accompany

the activation of ERK1/2 proteins (Bradford and Soltoff, 2002) and more recently has

been shown to induce the production of reactive oxygen species in murine

macrophages in a P2X7-dependent manner (Martel-Gallegos et al., 2010). If

extended to satellite cells, it may confer negative effects on mitochondrial energy

production, a phenomenon well documented in relation to altered Ca2+

homeostasis in

mdx muscle (Kuznetsov et al., 1998, Shkryl et al., 2009, Menazza et al., 2010).

Recently, over-expression of the endoplasmic reticulum Ca2+

ATPase pump SERCA

in both mdx and -sarcoglycan null mice has been shown to rescue the dystrophic

phenotype and confer normal mitochondrial morphology by a mechanism proposed to

be Ca2+

dependent (Goonasekera et al., 2011). Principally regarded as a pro-apoptotic

stimulus, PKCactivation has previously been shown to function in both pro- and

anti-apoptotic manners, activated through a caspase-3-mediated cleavage event

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(Allen-Petersen et al., 2010) that may relate to p38 activation prior to P2X7-

dependent large pore formation (Donnelly-Roberts et al., 2004). Whether a functional

link between caspase-3 mediated PKC cleavage and P2X7 activation exists to mirror

that of the P2X7-dependent caspase-1-mediated IL-1 cleavage and secretion

associated with the inflammatory response in macrophages and also known to operate

in myoblasts (Rawat et al., 2010) remains to be seen. Therefore, the up-regulated

phosphorylation of PKC seen here in dystrophic myoblasts treated with ATPe could

suggest that this signalling event may be linked to P2X7-induced ERK1/2 activation

in these cells. However, the functional implication of PKC activation in myoblasts

is unclear.

Perhaps then, a fine balance in P2X7 activation in satellite cells exists (where large

pore formation has been found to be absent despite the expression of pannexin-1)

between the activation of different members of the MAPK family proteins. Indeed,

activation of p38 has been shown to be sufficient to confer constitutive activation to

the quiescent satellite cell population (Jones et al., 2005). Therefore, the constitutive,

tonic level of P2X7 receptor activation observed in dystrophic myoblasts here may be

sufficient to trigger ERK1/2 and p38-mediated satellite cell activation and

proliferation via the JAK2 tyrosine kinase pathway, which itself is a mediator of

growth and proliferation in myeloid progenitor cells (Kovanen et al., 2000) and

myoblasts, acting via the STAT pathway (Wang et al., 2008). This constitutive P2X7

receptor activation may then extend to myofibres as well, owing to the retention of

P2X7 receptor in myotubes (Figure Appendix 3). In turn, constitutive activation of

the JAK-STAT pathway could inhibit MRF expression and terminal differentiation,

resulting in the retention of centrally nucleated myofibres; a hallmark of the pathology

in the mdx mouse (Yokota et al., 2006). However, the JAK1/STAT1/STAT3 and

JAK2/STAT2/STAT3 pathways have been shown to have opposing functions in this

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regard. The JAK1 pathway has been shown to repress MyoD and MEF2 expression

and the JAK2 pathway to enhance their expression, via unresolved mechanisms

(Wang et al., 2008).

Another partner in this cascade may be annexin A6, shown here to display

significantly higher phosphorylation status in dystrophic myoblasts in response to

ATPe stimulation. Its proposed role is as a negative effector of MAPK signalling and

Ca2+

channel function through translocation to the plasma membrane (Grewal et al.,

2005, Grewal et al., 2010) and it may be of significant relevance to the susceptibility

of dystrophic myoblasts to MAPK cascade induction. As eluded to in the chapter-

specific discussion, a particularly interesting property of the annexin A6 response in

lymphocytes is its Ca2+

dependent translocation to membrane associated vesicles,

proposed to play a functional role in inflammatory mediator release (Podszywalow-

Bartnicka et al., 2007). It would be of significant relevance in relation to not only the

proliferation of satellite cells, but also to the establishment of the immune response

characteristic for the mdx muscle pathology. Indeed, Rawat‟s group have recently

highlighted the potential role of toll-like receptors (TLR-2 and TLR-4) in mediating

P2X7-dependent IL-1 release in myoblasts derived from the dysferlin null mouse in

response to stimulation with lipopolysaccharide (LPS) and BzATP (Rawat et al.,

2010). This response is more typical for macrophages than skeletal muscle cells, a

fact which poses two questions: What role can LPS play in the co-stimulation of

P2X7 in dystrophic muscle; and what is the functional link bringing together P2X7

receptors, TLRs and dystrophin expression in skeletal muscle? On the first point,

although sources of LPS are widespread, no significant roles have been documented

for pathogen-mediated responses in dystrophic muscle. However, LPS is just one

example of molecules that bind to TLRs and P2X7 receptors and activate the

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inflammasome pathway. Amongst those are endogenous danger/alarm signals, eg.

HMGB1, S100 and some other less characterised proteins. These molecules present in

muscle may be the stimulus responsible for inflammatory cascade induction in

dystrophic muscles.

10.7 Functional relevance of altered P2X7 expression in dystrophic muscle

and the potential for therapeutic intervention

In addressing the origins of P2X7 receptor up-regulation in mdx myoblasts, one

protein of particular interest is biglycan, a small leucine-rich proteoglycan (SLRP)

protein found sequestered in the extracellular matrix (EM) and secreted in soluble

form by activated macrophages (Schaefer et al., 2005). There it complexes with

TLR2, TLR4 and P2X7/P2X4 receptors to form a larger multimeric mediator of

inflammasome formation, driving caspase-1-dependent IL-1 processing and

secretion (Babelova et al., 2009). Biglycan null mice display mild dystrophic

phenotype, proposed to be a product of biglycan‟s role in tethering selected

components of the DAPC (Mercado et al., 2006, Brandt and Pedersen, 2010, Brandan

et al., 2008). Moreover, biglycan expression has been shown to be up-regulated in

mdx muscle (Bowe et al., 2000). Interestingly, it has been shown recently that

components of the DAPC are rescued by biglycan‟s recruitment of utrophin to the

sarcolemma, following the demonstration that systemically delivered recombinant

biglycan confers utrophin up-regulation and a reduction in pathology in the mdx

mouse (Amenta et al., 2011). This may illustrate a functional distinction between

soluble and membrane bound biglycan derivatives. If up-regulation of the membrane

bound biglycan protein confers therapeutic gain in the mdx mouse through partial

DAPC restoration, then perhaps the soluble protein secreted by macrophages has the

opposite effect, mediating the P2X7-dependent biglycan inflammasome response.

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Therefore, therapies aimed at inhibiting the soluble biglycan protein may also be of

therapeutic merit here. In support of this theory is the observation that biglycan has

recently been shown to be secreted by myoblasts and up-regulated in cells undergoing

differentiation (Henningsen et al., 2010), suggesting the possibility for a positive

feedback mechanism involving P2X7 receptor stimulation and biglycan secretion that

would be constitutively active, based on the results presented here. Hence the

secreted form of biglycan may have a detrimental function in mdx muscle, so far

masked by the therapeutic benefits conferred through delivery of the membrane-

bound form, and may be involved in potentiating the immune system response of

dystrophic muscle. However, additional complexities can be envisaged, when

considering the role of other cell types. For example, biopsied fibroblasts have been

shown to be one of the main sources of biglycan in DMD muscle (Fadic et al., 2006).

Indeed, TLR4 expression in myoblasts is induced by IL-6 via STAT3 (Kim et al.,

2011), hence TLR4 expression and biglycan recruitment could be P2X7-dependent in

myoblasts based on the ERK1/2 activation data presented here. Moreover, IL-6

secretion is induced by IL-1 in a dose dependent manner in myoblasts (Gallucci and

Matzinger, 2001, Gallucci et al., 1998) and IL-1 is itself processed and released in a

P2X7-dependent manner in both myoblasts and macrophages (Rawat et al., 2010,

Babelova et al., 2009, Ferrari et al., 1997, Di Virgilio, 2007). Thus, heightened P2X7

receptor expression and activation, inducing apparently self perpetuating processing

and release of IL-1and IL-6 may serve to unify several functional and indeed

morphological pathological hallmarks of mdx muscle. Moreover, IL-6 secretion can

also be P2X7-dependent in fibroblasts (Solini et al., 1999), suggesting the potential

for contributions from other cell types. Additionally, subpopulations of macrophages

have been described to contribute different functional effects in regenerating

dystrophic muscle. A CD68+ M1 population is involved in the pro-inflammatory

tissue damaging response, and a CD163+/ CD206

+ M2 population, further divided

into M2a, M2b and M2c, dependent on their response to IL-4, IL-6 and IL-10

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(Villalta et al., 2009, Tidball and Villalta, 2010). Therapeutic benefits have been

documented using IL-10 mediated inhibition of M1 macrophage activation in mdx

muscle (Villalta et al., 2010), suggesting the possibility for similar manipulation of

IL-6 induced signaling cascades. However, the demonstration of heightened myoblast

differentiation responses to IL-10 in the same study eludes to the intricacy of crosstalk

existing between myoblasts, macrophages and probably fibroblasts, where elucidation

of proper distinction between so called „myokine‟ (Brandt and Pedersen, 2010,

Pedersen, 2010) and cytokine signaling must first be addressed, so as to alleviate the

possibility of inadvertently down-regulating potentially beneficial regenerative

effects.

Finally, this study has shown P2X7 receptor expression to be retained following the

differentiation of myoblasts to myotubes, suggesting a role for this receptor in both

satellite cells and contractile myofibres. Myofibre damage in DMD muscles has been

shown to result from increased [Ca2+

]i levels not attributable to sarcolemmal damage

(Millay et al., 2009). Additionally, abnormalities in Ca2+

influx via L-type, TRP and

stretch activated channels have been documented in mdx myotubes (Millay et al.,

2009, Vandebrouck et al., 2002, Kumar et al., 2004, Yeung et al., 2005, Friedrich et

al., 2004), and Ca2+

channel blockers have proved beneficial in delaying the

dystrophic pathology in limb muscles (Jorgensen et al., 2011). The data presented in

this study suggests that inhibitors of ATP receptor-mediated Ca2+

influx may be

beneficial in reducing the severity or slowing the onset of the DMD pathology.

Indeed, a broad P2 receptor antagonist has previously been shown to confer

therapeutic benefit in mdx mice (Iwata et al., 2007).

174

10.8 Conclusion

Current research into therapeutic intervention in DMD relate largely to vector

borne gene therapy and stem cell based approaches, and although much has been

learned, problems of low transgene expression, immune rejection, low proliferative

and migratory potentials and difficulties in assessing expression/migration levels are

continually addressed in the literature (Arechavala-Gomeza et al., 2010, Meng et al.,

2011, Meng et al., 2010, Morgan et al., 2010, Mendell et al., 2010, Trollet et al.,

2009). Complimentary to these ideas, this study has identified a specific ATP

receptor abnormality in skeletal muscles of the adult mdx mouse. With up-regulated

ATP receptor expression and high levels of immune cell invasion, the arena of

dystrophic muscle may become ever more inviting to the purinergic investigator;

offering a novel and in many ways unique in vivo opportunity to study the entire

nucleotide receptor family in its full glory. P2X7 has been shown here to be integral

to the phenotype of the mdx mouse, representing an extension of previously known

mechanisms of purinergic signalling in myoblasts and highlighting a novel role for

P2X7 in dystrophic muscle. It is proposed that this receptor may be a candidate for

therapeutic evaluation, and indeed may offer some insight into possible ways in which

myoblasts can be affected by the multifaceted Ca2+

abnormalities of dystrophic

muscle.

175

11

Further studies

11.1 Analysis of P2X7 splice variant expression in mdx/P2X7 double

knockout mouse models.

Complementary to the idea of pharmacological in vivo P2X7 receptor

blockade in the mdx mouse, is the notion of generating mdx/P2X7 double knockout

(DKO) animals for analysis of the effect of complete P2X7 receptor expression

ablation on the dystrophic pathology in these animals, thus subverting those problems

associated with antagonist selectivities, dosages, injection courses and methodologies.

To date, two P2X7 KO mouse models are available to the scientific community: one

generated by GlaxoSmithkline Ltd. (GSK) and the other by Pfizer Ltd. Both have

invested in the generation of P2X7 KO mouse models following the intense interest in

the receptor over recent years. Both these knockouts are available to our laboratory.

DKO animals were genotyped using PCR based analysis of genomic DNA extracted

from ear clips taken at 2 weeks of age. Specific regions of DNA were amplified using

primer sets able to differentiate the single nucleotide mutation in exon 23 of the mdx

strain from the WT sequence (Amalfitano and Chamberlain, 1996) as previously

described (see Figure 3.2.1), which would be absent in homozygous mdx animals.

Other primer sets were designed that were able to differentiate P2X7 KO from WT

alleles based on overlapping sequences of the lacZ and neomycin gene cassettes

inserted during the generation of the GSK and Pfizer P2X7 KO animals respectively

(Solle et al., 2001, Sikora et al., 1999).

176

The recently described P2X7k variant (Nicke et al., 2009) was found to escape

inactivation in both the GSK P2X7 KO and mdx/P2X7 DKO animals models (Figure

11.1; A), and indeed a similar situation was observed for the currently un-published

P2X7c variant, which escaped inactivation in the Pfizer P2X7 KO animal through a

similar mechanism (Figure 11.1; B) – although semi-quantitative PCR reproducibly

demonstrated much lower levels of this variant in the Pfizer P2X7 KO than were

consistently found for P2X7k variant expression in the Glaxo P2X7 KO animal.

Even the low level expression of P2X7 splice variants in DKO animals

suggested here may be sufficient to mask any beneficial effects of silencing this

receptor against the mdx genetic background, yet these animals may still prove useful

- with the diversity of proposed conditions and pathways involving the P2X7 receptor,

the existence of splice variant specific KO animals may allow variant specific

functions to be more clearly defined. Data available to date concerning any derivation

of function from P2X7 KO animals must be treated with some caution. The general

consensus in the P2X7 field has been that the available P2X7 antibodies were

unreliable based on their signal retention in the KO animal models (Sim et al., 2004),

however, data collected by our lab and Murrell-Lagnado‟s group (Cambridge),

suggests that effects previously attributed to antibody-specificity are actually the

result of novel splice variant expression patterns (manuscript in preparation).

Although P2X7 pharmacological profiles have been classically documented using

HEK cells or oocytes, due to the technical ease of transfection and patching,

respectively, it would none the less be interesting to characterise the expression and

function of certain variants in myoblasts derived from Glaxo- and Pfizer-derived KOs

and DKOs; the links this study has suggested between P2X7 and ERK1/2 activation

consist of bi-fold responses, where ERK1/2 activation can lead to both survival and

growth or apoptosis and cell death by as yet little understood mechanisms.

177

Solutions to the problem of splice variant expression in the current KOs might

include: The creation of a true P2X7 KO using homologous recombination to excise

the entire gene in embryonic stem cells prior to implantation and generation of a new

KO line. Alternatively, other informative methodologies might involve the generation

of a P2X7 over-expressing strain, possibly in an inducible and tissue-specific manor,

or alternatively to biopsy normal C57 muscle, transfect the derived primary myoblasts

with P2X7 under viral promoter control and transplant the cells back into the normal

mouse to determine whether the increased purinergic sensitivity would be beneficial

or detrimental to the overall muscle pathology.

Homozygous DKOs were obtained in the F3 and F4 generations of GSK and

Pfizer derived litters respectively, as confirmed by PCR analysis using primer sets

designed to amplify sequences of gDNA specific to WT or KO variants of mdx, GSK

P2X7 KO and Pfizer P2X7 KO animals. For P2X7 variants, PCR products could be

visualised together due to differences in product size, whereas mdx variants were

visualised separately since the mdx mutation comprises a single base pair mutation,

which confers no difference in PCR product size. Amplification of KO specific

sequences combined with absence of WT sequence amplification for both dystrophin

and P2X7 was used as confirmation of homozygosity in the generation of mdx/P2X7

DKO animals. Characterisation of both DKO animals is ongoing.

178

Figure 11.1. P2X7 splice variants escape inactivation in P2X7 KO animals. RT-PCR

demonstrated the expression of P2X7k variant in tissues of the GlaxoSmithKline (GSK)

P2X7 KO mouse and mdx/GSK P2X7 DKO mouse (A; lower panel). Expression of P2X7c

variant was detected in tissues of the Pfizer P2X7 KO mouse (B; lower panel). Upper panels

in A and B illustrate the design of KO constructs.

179

12

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13

Appendices

Work presented in these appendices relates to experiments carried out by the

author himself, by a colleague, Dr. Wotjek Brutkowski, and by a collaborator, Mr

Morten Ritso (University of Newcastle). That presented here (with permission) aims

to complement and clarify certain statements that appear in the main thesis text.

Figure Appendix 1. Desmin staining of wild-type/mdx immortalised myoblast cultures.

Immunoflourescence images showing tmie course of desmin (green) expression in

differentiating H-2Kb-tsA58 and H-2Kb-tsA58/mdx-derived myoblast lines over 6 days

(upper panels). 488c denotes secondary antibody staining only with primary omitted (lower

panels; negative control). Nuclei are counterstained using DAPI (blue).

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Figure Appendix 2. Annexin V staining of ATPe treated wild-type/mdx immortalised

myoblasts. Flow cytommetry-derived plots depicting percentage gatings following ATPe

treatment of H-2Kb-tsA58 (right panels) and H-2Kb-tsA58/mdx (left panels) immortalised

myoblasts. Apoptosis (assayed for through Annexin V translocation to the outer membrane

leaflet) was not observed in either genotype following treatment with ATPe (500M; 10 mins)

or Staurosporine (2M; 4 hours).

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Figure Appendix 3. Up-regulations in P2X7 receptor expression in dystrophic

myoblasts are retained following differentiation. Representative immunoblot analysis of

P2X7 receptor expression in H-2Kb-tsA58 and H-2Kb-tsA58/mdx-derived cultured myotubes

following 8 days in differentiation media.

Figure Appendix 4. Pannexin-1 is expressed in cultures of wild-type and mdx

immortalised myoblasts and myotubes. RT-PCR showing Pannexin-1 expression in H-

2Kb-tsA58 and H-2Kb-tsA58/mdx-derived myoblasts (mb) and 8 day differentiated myotubes

(mt). GAPDH represents a positive control.