EP2425256A1 - Method for diagnosing and treating muscular dystrophy - Google Patents

Abstract

The invention describes a method for identifying an individual exhibiting symptoms of, or having a propensity to develop muscular dystrophy comprising determining the level of expression in a tissue sample from the individual of one or more proteins selected from pax-7, caveolin-3 and/or fast-myosin. The use of caveolin-3 to treat muscular dystrophy and compositions containing the compound are also claimed.

Description

METHOD FOR DIAGNOSING AND TREATING MUSCULAR DYSTROPHY

Assay

The invention relates to methods for identifying individuals with, or who are likely to develop, muscular dystrophies, such as Duchenne Muscular Dystrophy (DMD) and limb girdle muscular dystrophy (LGMD), and to assay kits.

Duchenne Muscular Dystrophy (DMD) is the severest a family of debilitating congenital diseases associated with disruption of function of the trans-sarcolemmal, dystrophin- glycoprotein complex (DGC). Classic DMD, which affects the entire skeletal musculature, arises from loss of an essential intracellular component of the DGC, dystrophin (Hoffman et al., 1987). The condition is characterised by early (~3 years age) and progressive disruption of skeletal muscle function leading to extensive muscle damage and causing early death (usually 20's). Death commonly results from complications arising from failure of respiratory muscles, however, 95% of patients with DMD develop a cardiomyopathy which, in 10-30% of cases is the primary cause of death (Cox and Kunkel, 1997). The mdx mouse, a dystrophin deficient model of DMD, exhibits many of the post-natal features of DMD including cardiomyopathy; skeletal muscular dystrophy associated with bouts of fibre regeneration; fibrosis; hyperproliferation and apoptosis of skeletal muscle myoblasts (Harper et al., 2002; Smith et al., 1995). The phenotype is less severe than human DMD although mdx life span is curtailed with respect to WT (Chamberlain et al., 2007). Loss of caveolin-3 results in a milder form of MD, LGMD-Ic, in which affected muscle groups are predominantly limb girdle and heart; caveolin-3 deficient mice (cαv-5^v) exhibit LGMD, T-tubule defects, cardiomyopathy and skeletal muscle apoptosis (Hagiwara et al., 2000; Minetti et al., 2002).

Embryonic skeletal muscles originate from the dermamyotomal region of the somites (Cossu et al., 1996; Hollway and Currie, 2003). In the chick embryo all regions of the dermamyotome appear to contribute myogenic precursors to this process in a time dependent manner (Gros et al., 2004). Dystrophin is first expressed in embryonic somite from E9.5, and could thus have a function in early myogenesis (Huang et al., 2000; Ilsley et al., 2002). Under control of inducing factors secreted by surrounding tissues, the myotome produces cells which become the (myf-5⁺) stem cell populations which, respectively, generate (from E10.5) the epaxial (deep muscles of the back) and (from El 1.5) the hypaxial (limb, abdominal, diaphragm) muscles (reviewed in (Hollway and Currie, 2003)).

In mammalian embryonic skeletal muscle differentiation there are thought to be two myogeneic waves generating the hypaxial lineage to produce a scaffold of primary myotubes and subsequently, large numbers of secondary myotubes, which comprise the bulk of newly formed muscle (Cossu et al., 1996). Secondary myotubes are a morphologically distinct subset of myotubes, longer and thinner than primary myotubes which form in clusters around a single larger primary myotube (Cho et al., 1994).

Myotube differentiation is dependent on the presence of functional muscle stem cell populations. Myf-5 is expressed in the somite, the earliest of the myogenic regulatory factors (MRF) at E8.5, it is the only MRF found prior to muscle differentiation and persists in all embryonic muscle groups throughout both primary and secondary myogenesis (Cossu et al., 1996). It is therefore a good marker for the embryonic myoblast population. Lineage analysis suggests that a majority of satellite cells derive from the somite (Armand et al., 1983). Extensive evidence now links the pax-7+ cell population in particular, as being essential to the correct functioning of the adult satellite cell population and particularly of its repair function (reviewed in (Zammit et al., 2006)) Pax-7 is a transcription factor, a repressor of myogenesis, and plays important roles in the maintenance and specification of the adult skeletal muscle stem cell population (the 'satellite' cell) and is expressed in undifferentiated muscle stem cells emerging from the somite from El 1.5 (Merrick et al., 2007; Relaix et al., 2006; Seale et al., 2000).

The specificity of function of individual skeletal muscle groups depends on the appropriate localisation of fast myosin isoforms to different myotubes; a process initiated during the late stages of gestation (Merrick et al., 2007). In mammalian embryos developmental (embryonic and neonatal) myosin isoforms are co-expressed in newly formed secondary myotubes with adult fast myosin isoforms (Cho et al., 1994). In later stages developmental myosin isoforms are down-regulated and replaced by adult myosin heavy chain (MyHC) isoforms in a muscle specific pattern, a process not completed until several weeks after birth (Agbulut et al., 2003; Merrick et al., 2007). The embryonic heart expresses two myosin isoforms (cardiac myosin α and slow/cardiac myosin β) in a temporally regulated manner. Mutation of β-cardiac myosin is implicated in cardiomyopathy (Geisterfer-Lowrance et al., 1990).

Dystrophin associates with the beta subunit of dystroglycan on the intracellular side of the sarcolemma and is an essential component of the DGC, a multifunctional protein complex, which links the extracellular matrix to the actin cytoskeleton in skeletal muscle myotubes (Ervasti and Campbell, 1993). In post-natal muscle, dystrophin deficiency results in complete breakdown of the DGC and the secondary down-regulation of a majority of the DGC proteins (Ohlendieck et al., 1993). Caveolin-3 localises both to skeletal muscle caveolae and to the DGC where, by means of a specific WW domain, it binds the same PPXY motif in the β- dystroglycan c-terminus that recognises and binds dystrophin, thus blocking the interaction between dystrophin and β-dystroglycan (Jung et al., 1995; Sotgia et al., 2000).

The competitive interaction between caveolin-3 and dystrophin for the β-dystroglycan binding site may be critical for some aspects of muscle development but this has not been extensively studied. All three genes are expressed early in development in chick, mouse, zebra fish and Xenopus laevis embryos (Biederer et al., 2000; Houzelstein et al., 1992; Nixon et al., 2005; Razani et al., 2002; Shin et al., 2003; Anderson et al, 2007). In zebra fish, loss of any of these proteins has been shown to disrupt myogenesis and cause gross muscle abnormalities, whilst in mouse, dystroglycan deficiency leads to early embryonic lethality (Bassett et al., 2003; Nixon et al., 2005; Parsons et al., 2002; Williamson et al., 1997).

The inventors have examined the impact of loss of caveolin-3 and dystrophin on skeletal muscle development in order to establish a functional role for these two proteins during myogenesis and have identified an assay for the early diagnosis of muscular dystrophy.

The inventors have found that examination of embryonic myogenesis of two distinct but functionally related, skeletal muscle dystrophy mutants (mdx and cav-3^';') establishes for the first time that key elements of the pathology of DMD and LGMD type 1C originate in disruption of the embryonic cardiac and skeletal muscle patterning processes. Disruption of myogenesis occurs earlier in mdx than cav-3^"'^' consistent with the milder phenotype of LGMD-Ic and the earlier (E9.5) expression of dystrophin. Myogenesis is severely disrupted in mdx embryos with developmental delay, myotube morphology and displacement defects, and aberrant stem cell behaviour. Caveolin-3 protein is elevated in mdx embryos. Both cav-3^^' (from El 5.5) and mdx (from El 1.5) exhibit hyperproliferation and apoptosis of myf- 5+ embryonic myoblasts, attrition of pax-7+ myoblasts in situ and depletion of total pax-7 protein in late gestation. Both have cardiac defects. In cav-3^'A there is a more restricted phenotype comprising hypaxial muscle defects, excess, malformed hypertrophic myotubes, 2- fold increase in myonuclei and reduced fast myosin heaving chain (FMyHC) content. Several mdx embryo pathologies including myotube hypotrophy, reduced myotube numbers and increased FMyHC have reciprocity with cav-3^'A . In double mutant (mdxcav^+/~) embryos deficient in dystrophin (mdx) and heterozygous for caveolin-3 (cav-3^+/~), caveolin-3 is reduced to 50% of WT and these phenotypes are severely exacerbated; intercostal muscle fibre density is reduced by 71% and pax-7 positive cells are entirely depleted from lower limbs and severely attenuated elsewhere; data which suggest a compensatory rather than a contributory role for the elevated caveolin-3 levels found in mdx embryos. These data establish a key role for dystrophin in early muscle formation and demonstrate that caveolin-3 and dystrophin are essential for correct fibre-type specification and emergent stem cell function. These data plug a significant gap in the natural history of MD and is invaluable in establishing earlier diagnosis for DMD/LGMD and in designing earlier treatment protocols leading to better clinical outcome for these patients.

Previous assays for Duchenne muscular dystrophy have involved looking for polymorphisms in the dystrophin gene. The gene is large and testing for all polymorphisms is not carried out. For example, testing 19 mutations only identified 65% of cases. This is currently only carried out when there is a family history of the disease. However, approximately a third of all cases are de novo arising spontaneously in an individual.

The test identified by the inventors is not based on polymorphisms in the dystrophin gene and potentially is applicable to all forms of muscular dystrophy. Moreover, the test is applicable to babies both before and after birth. More traditional forms of diagnosis are not carried out until the child reaches 3-5 years of age. The assay allows the early diagnosis of the disease which allows early treatment of that disease, for example, by replacement of pax-7 positive stem cells.

Pax-7/Myf5 cells stem cells have been suggested to be used in therapeutic uses (WO 2007/059612). Such cells are suggested to be used as myogenic progenitor cells for the treatment of muscle diseases including muscular dystrophies. The cells are used as a source of new muscle cells for the treatment of diseases. However, there is no suggestion of using Pax-7 as a marker for a muscular dystrophy. The protein is simply used as one of two markers to identify a sub-population of stem cells to be used.

The invention provides a method for identifying an individual exhibiting symptoms of, monitoring the treatment of or progression of, or having a propensity to develop symptoms of, a muscular dystrophy comprising determining the level of expression in a tissue sample from the patient, of one or more proteins selected from pax-7, caveolin-3 and/or fast-myosin.

The method may be used to identify individuals exhibiting symptoms of or having a propensity to develop symptoms of the disease.

When treatment or progression is monitored, the level in the sample may be compared to a level in a sample from the individual taken previously, such as days, weeks or months previously.

The level of expression of the or each protein may be compared to a pre-determined level associated with, for example, an individual not having symptoms of, or having a propensity to develop, a muscular dystrophy, and/or compared to a pre-determined level that has previously been identified as being associated with individual exhibiting or having a propensity to develop, the muscular dystrophy.

Pax-7 and caveolin-3 have been identified as having particular relevance in the early determination of whether, for example, a baby or pre-term baby is likely to develop a muscular dystrophy. Preferably one or both of pax-7 and/or caveolin-3 is measured. Pax-7 may be measured. Caveolin-3 may be measured.

Pax-7 and/or caveolin-3 may be measured they may be measured with or without measuring fast-myosin.

These two markers (pax-7 and caveolin-3) in particular allow the early identification of the potential for the individual to develop muscular dystrophy. Prior to this, it has been necessary to await the development of the disease, which is often not recognised until the individual is several years old. Alternatively, there are a number of prior art assays which look for specific polymorphisms in, for example, the dystrophin gene. However, such assays are typically for the polymorphism associated with the particular family, where there is a family history of such polymorphisms. Moreover, the accuracy of such assays typically remains low.

Accordingly, the tissue sample may be obtained from an individual before or after the birth of the individual. Myogenesis typically occurs from 8-9 weeks of a pregnancy. Hence, potentially samples could be taken from that stage onwards. The individual may be, for example, a child for example, less than 16, less than 12, less than 10, less than 6, less than 3, less than 2 or less than 1 year old, and may be still within the womb. The individual patient may be between birth and 3 or 6 months old.

Muscular dystrophy (MD) refers to a group of genetic, hereditary muscle diseases that weaken muscles in mammals, such as humans. MDs are usually characterised by progressive skeletal muscle weakness, defects in muscle proteins and death of muscle cells and tissues. Preferred diseases include Duchenne (DMD), limb girdle (LGMD), Emery-Dreyfuss, oculopharyngeal, distal, myotonic, congenital, Becker, and/or facioscapulotumeral. The disease may be DMD and/or LGMD.

Typically, a decrease in expression of pax-7 or altered expression of caveolin-3, compared to a normal individual, is indicative of the individual exhibiting symptoms of, or having a propensity to develop symptoms of, a muscular dystrophy.

Whilst the individual may be a human individual, there are a number of different animals which also develop muscular dystrophy-like symptoms. Such animals are typically mammals, and may include, mice, rats, sheep, dogs, horses, cats and non-human primates.

Fast-myosin may be measured. The inventors have determined that fast-myosin has different expression levels in different diseases. For example, it is increased in the mouse model for Duchenne muscular dystrophy, but decreased in limb girdle muscular dystrophy. Hence, it is believed that assaying for the levels of expression of that protein gives an indication of the likely outcome of the disease. Preferably fast-myosin is assayed in combination with an assay for one or both of pax-7 and/or caveolin-3. Fast-myosin may also be used alone to diagnose, for example, particular types of muscular dystrophy such as DMD and LGMD.

The assay may additionally comprise determining the level of expression of insulin-like growth factor-2 (Igf-2) in the tissue sample. Igf-2 is over expressed in muscular dystrophies such as Duchenne muscular dystrophy, and reduced in caveolin-3 deficient LGMD. Again this gives an indication of the prognosis of the disease.

Moreover, the following gene products downstream of Igf-2 may be assayed: CDKNlc/P57kip2 and/or pAkt.

Igf-2, CDKNl c/P57kip2 and/or pAkt may also be assayed separately to the other proteins. That is, for example, Igf-2 may be assayed separately without measuring pax-7 and caveolin- 3. It may be used with CDKNlc/P57kip2 and/or pAkt.

Merrick D et al (BMC Develop. Biol. (2007), 7, page 65) disclosed that insulin-like growth factor-2 (Igf-2) is an embryonically expressed growth factor. This was known but its specific expression in skeletal muscle myotubes was not. The main finding of the paper was that Igf- 2 influences the ratio of fast myosin positive fibres.

All of the proteins described above are generally known in the art, with their protein and nucleic acid sequences encoding such proteins generally known in the art.

The tissue sample is typically a sample of muscle tissue, for example, in the form of a biopsy sample. However, it is believed that fast-myosin and caveolin-3, may also be found within the blood of individuals with muscular dystrophy, due to degeneration of the muscle fibres within the individual.

The protein expression may be determined by either measuring the level of protein expression directly, or indirectly by determining the level of mRNA in the sample.

Typically the expression level of the protein may be determined by immunoassay. Antibodies specific for the proteins described above are generally known in the art. Such antibodies may be labelled directly or indirectly by methods generally known in the art. For example, the sample of tissue may be immunostained with a suitably labelled antibody. The sample may be fixed prior to staining. The inventors have found that paraformaldehyde is particularly effective when used with antibodies specific for the proteins, and in particular for antibodies specific for pax-7. Hence preferably, the fixative used is paraformaldehyde. The binding of antibodies to the proteins may be determined by methods generally known in the art. For example, avidin-biotin methods are generally known in the art. Such assays typically use horse-radish peroxidase in combination with a substrate such as 3, 3', 5, 5' tetramethyl-benzidine to form a coloured product, which may then be visualised under, for example, a microscope. The inventors have found in particular, that tyramide signal amplification (TSA) produces especially good staining, for example when used with pax-7. Hence, preferably TSA is used in the immunostaining.

Alkaline phosphatase may also be used. This utilises a pre-formed cyclic enzyme anti- enzyme immunocomplex composed of three enzyme molecules (alkaline phosphatase) and two antibody molecules. The technique can be visualised by typically using a blue dye (Fast Blue BN) or a red dye (Fast Red TR).

The immunoassays may be visualised using a directly labelled anti-protein antibody. Alternatively, they may be visualised indirectly by using an anti-immunoglobulin antibody to bind the anti protein antibody. That secondary antibody may itself be labelled.

The or each antibody may be labelled with a fluorophore. Such fiuorphores are generally known in the art. They include, for example, fluoresceine, fiuoresceine isothiocyanate (FITC) and rhodamine. Such fiuorphores fluoresce at different colours. Hence, preferably the antibodies used in the assays (and indeed kits, defined below), have different fiuorophores and/or different methods of being visualised, for example with horse-radish peroxidase, to allow different proteins to be assayed from the same tissue sample.

The proteins may also be visualised by adding the labelled antibody to the tissue sample, partially purified sample or purified proteins obtained from the sample, to identify the total antibody bound to the sample. The proteins in the sample may be purified and visualised by immunoblotting. Alternatively, or additionally, the level of expression of the protein may be determined by identifying the level of mRNA expressed in this tissue sample. The level of mRNA expression may be determined by quantitative polymerase chain reaction (QPCR) or realtime PCR (RT-PCR). This technique utilises a pair of primers specific for the mRNA encoding the protein. Such techniques are generally well-known in the art.

There are a number of commercially available systems in the art which allow quantitative PCR, for example, using SYBR™ stains. Manufacturers include Applied Biosytems Limited and Promega Corporation.

The invention also provides a kit for use in a method according to the invention, comprising two or more antibodies or a pair of PCR primers specific for two or more of pax-7, caveolin-3 and/or fast-myosin, or mRNA encoding such proteins.

The protein may be pax-7 protein or mRNA encoding pax-7. Alternatively or additionally, the protein may be caveolin-3 or mRNA encoding caveolin-3.

The protein may additionally be fast-myosin or mRNA encoding fast-myosin.

The antibodies may be directly labelled with a suitable label, such as one of those described above. Such labels include fluorescent labels and enzyme-based labels such as alkaline phosphatase or horse radish peroxidase.

The kit may comprise an antibody for one protein and a pair of primers for a second protein. Alternatively, the kit may comprise PCR primers for two or more other proteins. The kit may comprise two or more antibodies for different proteins. In the latter situation, the antibodies may be labelled with different labels to allow substantially simultaneous measurement of the amount of each protein.

The kit may additionally comprise paraformaldehyde. Paraformaldehyde has been found to be particularly effective in allowing the fixation of muscle samples. This may be provided in combination with an antibody specific for pax-7 protein.

The kit may additionally comprise an antibody or PCR primer pair specific for Igf-2. Where antibodies are provided in the kit, then the kit may comprise one or more reagents for tyramide signal amplification. This may include, for example, biotinylated tyramide.

The kit according to the invention may comprise one or more fixatives for fixing the sample.

The kit may comprise instructions for use with the kit. The instructions may comprise details of typical levels of protein expression associated with, for example, an individual exhibiting symptoms or, or having a propensity to develop, muscular dystrophy. The instructions may comprise details of expression levels in normal individuals. This allows a comparison of the data obtained from the sample from the individual to be compared to pre-determined values in order to allow a practitioner to assess the validity of the data obtained. One or more controls with known concentrations of the proteins may be provided.

Where PCR primers are provided, then the kit may additionally comprise one or more labels or other reagents to allow quantitative PCR to be carried out. This may include, for example, SYBR™ green.

The kit may also comprise one or more control samples of protein at a pre-determined concentration for use a standards within the assay.

The results obtained by the inventors have also indicated that caveolin-3 may be used to alleviate symptoms of muscular dystrophy.

Accordingly, the invention provides a method of treating a muscular dystrophy comprising administering caveolin-3 to a patient or increasing caveolin-3 expression in a patient. Methods of increasing the expression of protein, or presenting protein in the treatment of muscular dystrophy have previously been demonstrated for utrophin. It is expected that caveolin-3 may be used in a similar manner.

Utrophin has been demonstrated to be useful for treatment of muscular dystrophies. For example, US 2009/054327, US 2008/160108 and WO 97122696 (incorporated herein by reference in their entirety), disclose ways in which the protein has been used in such treatment. Pharmaceutical formulations comprising caveolin-3 in combination with a pharmaceutically acceptable carrier are also provided. The invention also provides caveolin-3 for use to treat muscular dystrophy.

The invention will now be described by way of example only with reference to the following figures:

Figure 1

(A) Skeletal myotube branching in mdx myotubes: (IC) intercostal, (L) limb, (F) facial muscle groups; 0: no abnormalities detected. (B) myotube branching, El 5.5 mdx biceps. Insert, enlarged section. Stain: haematoxylin and eosin. (C-H) Pan-myosin (MF20), proximal muscle, green bars show WT myotube width (C,F) WT; (D,G) mdx, hypotrophic myotubes, black arrow myotube splitting; (EJX) cav-3^'1', hypertrophic myotubes. (F-H) Variable myotube diameters in (G) mdx and (H) cav-3^''^' muscle fibres compared to (F) WT. (I) Displaced myotubes (combined tangential and misaligned myotubes) as a proportion of total counted. 0: no displaced fibres (J) Displaced myotube scoring strategy: tangential (T) > 25°, misaligned (M) < 25° from the median. (K) Proportion of misaligned mdx myotubes at E 13.5. (L-N) E 13.5 IC muscles, MF20; (M) mdx misaligned myotubes (black arrow, M) no myotube displacement in (L) WT (N) cav-3-/-. Error bars: s.d. Size bars 20 micrometers. Student's t- test; * p < 0.01; ** p< 0.001, compared to WT value.

Figure 2

(A-F, I-N) mid-section hearts immunostained for β-cardiac myosin heavy chain (N2.261) El 3.5 (A) WT; (B) mdx; (C) cav-3^'/- showing (B) mild and (C) extensive, ventricular wall hypertrophy, Compared to (D) WT, E 14.5 ventricle apex structure is disorganised in cav-3^{' '} (E) and less well developed in mdx (F). (G-H) MF20 stained El 5.5 cav-3^''^' heart (G) Disorganisation of the ventricular wall, (H)^"cardiac myocytes criss-cross each other. (I-J) El 4.5 atrial trabeculae (tb) are (I) short and stubby in cav-3^''^' and (J) hookshaped in mdx. (K- P) E17.5 atrium; (L-P) attenuated N2.261 labelling and distension of cell layers in (L, O) cav-3^''^' trabeculae (tb) and (M, P) mdx atrial wall, compared to (K, N) WT. Size bars 20 micrometers. (Q) low magnification image of MF20 labelled mdx heart to illustrate the orientation and matching of hearts sectioned sagitally through the most central portion of the heart.

Figure 3

Arrangement of myonuclei in E15.5 proximal muscle: (A) WT, helical, even spacing; (B) mdx, central location; (C) cav-3^'1' close central location, irregular spacing. Overlaid white stars (*), myonuclei in one myotube; small white arrows, myonuclei. In WT (A) small white arrows indicate peripheral nuclei in two separate myotubes. Peripheral nuclei are not evident in mdx and cav-3^''^' muscles (B-C). (D) Bunching of myonuclei at myotube ends, cav-3 ^''^'. (E- G) transverse sections of E15.5 WT (E), mdx (F) and (G) cav-3^'1' lower proximal limb myotubes showing peripheral myonuclei (arrow) are associated with WT but not dystrophic embryo myotubes at this stage. The hypotrophy of mdx and hypertrophy of cav-3 -/- myotubes can also be seen; green line indicates WT myotube diameter. (H) WT, (I) mdx and (J) cav-3^''^' lower magnification image of lower proximal limb region showing the reduced fibre density of mdx and increased density of cav-3^''^' muscle fibres compared to WT in matched embryo sections. (K) Myonuclei numbers are doubled in cav 3 ^" (E13.5-E17.5) compared to WT; * p < 0.05. Slight (not statistically significant) reduction in mdx myonuclei (E13.5-E17.5) Mean myonuclei content of myotubes is constant between E13.5-17.5. (L) Increase in cav-3^''^' (E13.5-E17.5) and decrease in mdx (E13.5) myotube density compared to WT. Myotubes counted over a fixed matched muscle area for each strain and stage, * p< 0.05. Size bars 20 micrometers.

Figure 4

(A) Outgrowth rate of embryonic myoblasts from muscle explant cultures is increased in mdx from El 1.5 and in cav-3^'1' at E15.5-E17.5, compared to WT explants cultured in parallel. (B) Myf-5 immunostained explant. (C) Hyperproliferation of embryonic myoblasts in mdx from El 1.5 and cav-3^''^' from E15.5, determined by Ki67+ immunoreactivity (D). (E) Elevated apoptosis from El 1.5 in mdx and E15.5 in cavS^''^' (F) Dapi staining, arrow indicates apoptotic cell. * p < 0.05 from WT; ** p < 0.01 from WT; # p < 0.05 between mdx and cav-3^' '^'. (G-H) El 5.5 primary cultured WT embryonic myoblasts (G) Myf-5 staining (H) second antibody control. (I) Outgrowth rate of El 1.5 WT explants increases (p < 0.05, *) in El 1.5 max explant conditioned medium {mdx CM) but not in cav-3^''^' or WT CM. Error bars, s.d. Figure 5

Pax 7 immunostaining in WT, mdx, cav-3 ^~A and mdxcav^+/~ lower proximal limb; (A-F, MN, QR) E15.5, (G-L) E17.5. (A-E; G-K) low magnification; (B-F; H-L) high magnification; size bars 10 micrometers. Q-R whole region view of WT (Q) and mdxcav-3^+/~ lower proximal limb. Putative apoptotic (fragmented) pax-7⁺ nuclei in mdxcav^+/~, cav-3^'1' and mdx but not WT (B, D & F) at E15.5 and (L) in mdx at E17.5 (black arrow). Attenuation of pax-7 staining, white arrows in cav-3^''^' and mdxcav-3^+/- (CD & MN) El 5.5; (I-J) El 7.5. (M-R). At El 7.5 mdxcav-3^+/~ lower proximal limb muscles are devoid of pax-7+ cells (O-P). (S) Immunoblotting, (i-iii) caveolin-3, (iv) pax-7. Exposure (i) 10 s; (ii)l min; (iii) 4 min. (iv) pax-7 protein is reduced in E15.5 & E17.5 dystrophic embryos. In a 4 minute exposure pax-7 is detected only in WT at El 1.5. (T) Densitometric analysis, caveolin-3 :α-tubulin ratio confirms increased caveolin-3 in mdx. * p < 0.05;. (U) Table showing the estimated fold increase of caveolin-3 in mdx (over WT) following separate analysis of 3 embryonic stages (4 experiments per stage), p = 0.05 for all stages. (V) Densitometric analysis, pax-7:α-tubulin demonstrating reduction of pax-7 protein in cav-3^'1' and mdx (E15.5-E17.5). E13.5 cav-3-/- embryos contain significantly more pax-7 than WT. * p < 0.05; (T, V) mean + s.d. of two separate experiments. (W) Immunoblot demonstrating that caveolin-3 protein is reduced in El 5.5 mdxcav-3^+/~ embryos (50% with respect to WT^#) and significantly reduced compared to El 5.5 mdx embryo siblings in which caveolin-3 is increased. Neomycin immunostaining confirms the presence of the cav-3KO transgene in cav-3^'1' and mdxcav-3^+l'. The reduced neomycin staining seen in mdxcav-3^+/~ embryos compared to cav-3^'A reflects their heterozygosity for the cav-3KO transgene. #densitometry: caveolin-3: α-tubulin ratio, track 1 WT compared to tracks 3 & 4, mdxcav-3^+/~.

Figure 6

(A-F) FMyHC immunostaining in El 3.5, (A-C) diaphragm; (D-F) intercostals. (A,D) WT, (B,E) mdx and (C,F) cav-3^'1' showing (B,E) reduced FMyHC in mdx and (C,F) increased FMyHC in cav-3^'1' respiratory muscles. (G-L) FMyHC in El 5.5 respiratory muscles (G-I) diaphragm; (J-L) intercostals (G,J) WT; (H,K) mdx, (I,L) cav-3^'1'. Increased FMyHC staining in (N) mdx and decreased FMyHC staining in (O) cav-3^'A compared to (M) WT E17.5 proximal limb. (P-S) In mdxcav-3^+/' FMyHC is also increased compared to WT at El 7.5 (Q) & (S) (proximal limb shown) and El 5.5 (R) (intercostal and diaphragm shown). Comparison of cav-3^"^^' (P) and (Q) mdxcav-3^+/' at E17.5 establishes that dystrophin deficiency suppresses the down-regulation of FMyHC seen in cav-3-/- and WT embryos. The excessive loss of FMyHC in El 7.5 cav-3^';" compared to WT embryos (M) can also be seen clearly (Q). (T) Immunoblotting of WT, cav-3^'A, and mdx shows attenuation of FMyHC in cav-3^'A and delayed production of FMyHC in mdx embryos compared to WT. (U) Densitometry, FMyHC: α-tubulin ratio demonstrates a statistically significant increase and reduction in FMyHC content in El 7.5 mdx and in cαv-J^"A embryos respectively. At El 3.5 there is a significant excess of FMyHC in cav-3^v~ embryos; mean + s.d. of two separate experiments. (V) FMyHC positive myotubes expressed as a proportion of total counted in WT, mdx and cav-3^''^' embryos (E13.5, E15.5 and E17.5). Error bars, s.d. * p < 0.05; ** p< 0.01. Size bars 20 micrometers.

Figure 7

El 7.5 (A-E) WT and (F-J) mdx mice hetero∑ygous for caveolin-3 (mdxcav-3^+/~), immunostained with pan-myosin (MF20) (B, E, G, J) or pax-7 (A, C-D & F, H-I) to illustrate the extensive loss of pax-7 myoblasts and muscle fibre density in the muscles of late gestation (El 7.5) embryos. (K-P) Higher magnification images of MF20 labelled intercostal muscle sections (matched 4^th intercostal for each embryo) showing the difference in myotube density between (K) WT, (L) mdx, (M) cav-3^"A and (N-P) three different mdxcav-3^+/~ E17.5 embryos. (R) fibre density assessed over a fixed grid area and (S) percent reduction in fibre density, in El 7.5 WT, mdx and mdxcav^+/~ (het) embryonic intercostal muscle. (T) WT and (U) mdxcav+/- E17.5 intercostal labelled with My32 antibody showing that almost all fibres are FMyHC positive. Note that fibres are downregulating FMyHC in many WT but not mdxcav-3^+/~ intercostal fibres.

Figure 8

Pax-7 human muscle biopsy:

Pax-7 immunostaining to identify pax-7+ skeletal muscle stem cells in an archival human juvenile muscle biopsy. Pax-7 labels cell nuclei. Dark staining indicates pax-7+ nuclei. Lighter staining is a Haematoxylin counterstain showing the location of pax-7 negative cell nuclei.

Figure 9 RT-PCR analysis (A) Densitometric analysis of pax-7 mRNA expression in six WT and six mdx mouse myoblast isolates establishes statistically significant suppression of Pax-7 expression in the dystrophic myoblasts. Data shown are mean and standard deviation of desitometry results individual myoblast isolates.

(B)RT-PCR showing detection of pax-& mRNA in six WT and six mdx myoblast isolates. Pax-7 expression in consistently suppressed compared to WT in dystrophic (mdx) myoblasts.

Figure 10

Igf-2 disrupted in MD mice

Embryonic skeletal muscle immunostained for the Insulin-like growth factor 2 (Igf-2) between stages E14.5 and E17.5. (A-D) Wild type (WT) muscles show a characteristic striped pattern of Igf-2 where 50% of fibres are Igf-2 immunostain positive. (E-H) dystrophin-deficient (mdx) muscles over-express Igf-2 between E14.5 and El 7.5 and loose the 'stripy' immunostain pattern of WT. In contrast (I-L) Igf-2 is lost in caveolin-3 deficient {cav-3^'1') muscles. (M) Quantification of immunstain pattern confirms these conclusions. (N) RT-PCR establishes that Igf-2 message (mRNA) is elevated in mdx and cav-3^^' embryos. The suppression of Igf-2 in caveolin deficient embryos is thus at the post-transcriptional level.

Figure 11

Genes downstream of Igf2

Genes downstream of Igf-2 (CDKN Ic/P57kip2 and pAkt) are also disrupted in dystrophic mice and could be included in a panel of genes (again for RT-PCR or immunodetection). See figure.

Level of CDKNlc/p57kip2 is significantly and reproducibly reduced in both dystrophin deficient and caveolin deficient mouse embryos compared to WT. (B) pAkt is elevated in mdx and suppressed in cav-3-/- mouse embryos.

Figure 12 shows depletion of Pax-7 myoblasts in human skeletal DMD. Figure 13 shows a graph showing depletion of Pax-7 myoblasts in human DMD and BMD compared to controls.

Figure 14 - Reducing caveolin- 3 levels to wild-type in mdx, increases the severity and prolongs the progression of dystrophic phenotype in mdx mouse muscle.

Figure 15 - The affect of Igf-2 on dystrophic myoblasts; Pax-7 and Pax-3 are up regulated by Igf-2 treatement. Increased caveolin-3 levels in dystrophic myoblasts is enhanced by Igf-2 treatment.

METHODS

Mouse models

Wild type (C57BL10) and isogenic mdx and cav-3^'1' mouse strains, were used, cav-3^'1' dystrophic mice on C57B110 background were from Yoshito Hagiwara (Tokyo) (Hagiwara et al., 2000). Mdx and C57BL10 were generated in house (Merrick et al., 2007). Double mutant mice (mdxcav+/-) were null for dystrophin (dys-/-) and heterozygous for caveolin-3 (cav-3+/- ) and were generated by intercrossing mdx and cav-3^*A using a strategy described previously to generate dystrophin deficient mutants heterozygous for an Igf-2 transgene (mdxIgf-2+/-; (Smith et al., 2000)). Genotyping was achieved by PCR for neomycin (to detect cav-3KO transgene) and caveolin-3 (expressed by WT, mdx and cav-3^+/~ but not cav-3^"A; Figure 7). Data shown are derived from 2 separate litters each of El 5.5 and El 7.5 embryos containing an approximately 50:50 ratio of mdx and mdxcav^+/~ embryos. Litter size (7-9 embryos per litter) was comparable to those obtained from WT and mdx.

Preparation of mouse embryos

Staged WT, cav-3^'A, mdx and mdxcav^+/~ embryos were fixed and processed for paraffin wax embedding as previously (Smith and Merrick, 2008). The morning of plug detection was estimated as E0.5. All sections were sagital (5μM). Plane and depth of cut were established at the midline of the embryo (bisection point) and using Kaufman's Atlas of embryology (Kaufman, 1992). Matched WT, cav-3^''^', mdx and mdxcav-3^+/~ sections were used for all analysis from at least three separate embryos per strain. Immunohistochemistry

Immunostaining and conditions for Igf-2, pan-myosin (MF20), fast-myosin heavy chain FMyHC (My32) and pax-7 antibodies are described previously (Merrick et al., 2007) immunostaining for CDNKlc/p57kip2 is also described previously (Westbury et al, 2001). pAkt deection was achieved at an optimized titre of 1/100 using an anti-pAkt rabbit polyclonal (No. 9277) obtained from New England Biolabs (UK) Ltd. An extended peroxidase blocking step was included in all staining runs. Second antibody controls showed no staining. B-cardiac myosin was detected using N2.261 antibody (1/1000; DHSB, Iowa City). CDNKl c/p57kip2, pAkt, IGF-2 and FMyHC staining was quantified as previously described forFMyHC and IGF-2 (Merrick et al., 2007) bu counting the proportion of antibody positive myotubes over a fixed grid area. Data were analyzed using Student's t-test and ANOVA. . At least 3000 myotubes were counted for each data point.

Pax-7 immunostaining was also carried out in archival juvenile human muscles biopsies (see Figure 8).

Analysis of myotube morphology and quantitation in MF20 stained sections

Sagital cut MF20 stained embryos from WT, cav-3^"'^" and mdx were matched for stage, plane and angle of section. To avoid artefacts the inventors counted only splits and branches entirely in the plane of cut, this was carefully controlled between sections. This analysis may underestimate splitting/branching events. Sagital sections from E13.5, E15. 5 and E17.5 embryos were carefully matched for location of morphological features against a standard published mouse atlas (Kaufman, 1995) at the midline point of the embryo to facilitate matching. Sections were counted blind and by two separate observers for both misalignment and branching phenotypes and subject to statistical analysis. Branching was scored over a fixed area using a grid graticule in the same longitudinally presenting muscles in the intercostals, upper and lower limb and facial muscle regions, for each mutant and WT embryo scored. Scoring of misaligned fibres was achieved by orienting the direction of the muscle fibres in longitudinal sections to a grid graticule and scoring over a fixed area for fibres which deviated by more than a 25 degree angle. We are confident our data are a reliable indicator of splitting/branching: when two experimenters counted slides, the second being unaware of the strain, splitting/branching was only identified in mdx and was statistically significant. For myotube counts we did not attempt to establish the ends of every myotube (an impossible task in sectioned embryos) but instead counted carefully matched sections of proximal, distal, intercostal and deep back muscles in 3 different embryos per strain over a fixed area (total myotubes counted per strain: 2-3000). This enabled us to estimate the proportion of myonuclei to myotubes and the average number of myotubes per section. Data analysis: ANOVA and Student's T-test.

Embryo explants

E11.5-E17.5 embryos were dissected to isolate areas rich in skeletal muscle cells. In all embryos head, spinal cord and internal organs were removed. In older embryos (E15.5-E17.5) skin and cartilage/bone were removed. Muscle-rich tissues were micro-dissected into micro- explants and cultured in microwells (Smith and Merrick, 2008; Smith and Schofield, 1994). WT, max or cav-37- El 1.5 explant conditioned medium (CM) was removed from confluent cultures, filtered (0.2 μm Acrodisc® Syringe filter; VWR International, UK), replenished with standard media supplements (20% FCS; 2mM Glutamine) and added to fresh El 1.5 WT explants for 18 days culture (Smith and Schofield, 1997). 180 explants from 3 embryos were analysed for each of the three strains included in this study. Data analysis by ANOVA.

Outgrowth analysis

Outgrowth is a reliable and highly reproducible measure of the growth rate of skeletal muscle explants (Smith and Schofield, 1994). Explants were cultured for 3 weeks and scored according to the level of confluence of cells in each well. Myf-5 immunostaining (Santa Cruz; titre 1/5000, Rabbit anti-Myf-5 c20) was used to establish the muscle origin of El 1.5- E17.5 WT, mdx and cav-3^~'^~ explant cultures; >85% of cells were myf-5+ (Smith and Merrick, 2008). Second antibody controls performed with each section were always negatively stained. Myf-5 c20 is used extensively to establish myogeneicity (Frock et al., 2006; Lindon et al., 1998).

Measurement of apoptosis and proliferation

Confluent explant cultures with myoblast morphological features were subcultured with dispase (Smith and Schofield, 1994), plated onto coverslips (5 X lO³ cells/cm²) for 6 hours before fixation. Apoptotic nuclei cells were stained with 10 ug/ml DAPI for 3 minutes (Smith et al., 1995). Proliferative cells were immunostained with Ki67 (Rabbit anti-Ki67, titre 1/1000: Novocastra Laboratories Ltd., UK) as above. For antigen retrieval (using a pressure cooker) coverslips were first firmly attached onto glass slides using standard paper clips. Three separate experiments were performed in duplicate for each strain (C57BL10, cavS^''^',

Isolation of protein and Immunoblotting

Protein was extracted from embryos directly into a glass homogeniser (1-5 ml; VWR International, UK) containing RIPA buffer. Immunoblotting was carried out using standard protocols and detected by ECL (Pierce Endogen Hyclone). Antibodies: fast myosin (My32, 1:1000); α-tubulin (1:1000, Sigma); pax-7 (1/1000); caveolin-3 (lΛOOO).Goat anti-mouse IgG-HRP (1:2000, Santa Cruz Biotechnology). Protein concentration was determined using an ELISA form of the Bradford assay (Merrick et al., 2007).

Pax-7 RT (reverse transcribed) real time PCR

Reverse transcribed (RT) PCR was carried out to quantitate the amount of pax-7 mRNA in myoblasts isolated from WT and mdx mouse skeletal muscle (Smith & Schofield, 1994). To obtain quantitative data linearisation and equalisation of the Pax-7 product were carried out as described for Igf-2 (Merrick et al, 2007). RT-PCR was carried out under these quantitative on six separate isolates of WT and six isolates of mdx myoblasts.

Polymerase Chain Reaction (PCR) cDNA samples were generated by the RT-PCR method described in Merrick et al, (2007) and subject to PCR in the following reagent mix:

• 5μl 5xPCR buffer (Promega).

• 1.5μl 25mM MgCl₂ (Promega).

• 0.5 μl 1OmM dNTP (Promega).

• 1 μl 25μM Primer mix 12.5μM of the forward and 12.5μM of the reverse primer (Alta Biosciences) in PCR dH₂O (Sigma).

• lμl cDNA.

• 0.1 μl Taq (Promega). - Final reagent added.

The Taq polymerase was added last in the PCR mix (as above), to prevent random transcription. The samples were loaded into a programmable thermal controller, which had been programmed specifically for the gene as follows: Pax7

Primer Sequences: pax7-forward: gct-acc-agt-aca-gcc-agt-atg

pax7 -reverse: gtc-act-aag-cat-ggg-tag-atg

Igf-2

Insulin-like growth factor 2 (Igf-2) has been previously reported. Staining methods and PCR methods are discussed in Merrick D et al (2007). Typically muscle biopsies were assayed.

Results

Branching, fibre misalignment and malformed myotubes characterise the embryonic phenotype of muscular dystrophy

WT, cαv 5^"A and mdx mice were immunostained with pan-myosin antibody (MF20) to reveal muscle fibre architecture (Fig. 1). Branching and fibre splitting is found in mdx epaxial (back), hypaxial (limb, respiratory muscles) and facial muscles from E13.5 to E17.5 and shows a dynamic temporal and spatial pattern consistent with it being an early event associated with myotube formation (Fig. IA- C and G). At matched stages and muscle groups mdx myotubes are hypotrophic, cav 3^~'^~ myotubes hypertrophic and myotube width varies more in dystrophic embryos with respect to WT (Fig. IC-E, green bars). Maximum morphological defects occur earlier in mdx intercostals (El 3.5) than proximal limb (El 5.5) consistent with later development of these muscles (Fig. IA). There is no fibre splitting in cav-3^~'^~ and WT embryonic myotubes (Fig. IA, C, E and F, H). Myotube alignment is also disrupted early in mdx myogenesis (Fig.ll). At E13.5 up to 1/8^Λ of mdx myotubes are displaced from the median fibre alignment in the proximal limb, intercostal and facial muscles (Fig. II). This is statistically different from E13.5 WT and cav-3^v~ muscle in which fewer than 5% of myotubes were displaced. When myotubes were divided into those which deviate more than 25° from the median (misalignment) and those displaced less than 25° (tangential fibres) more than half (7-9%) of displaced E 13.5 mdx myotubes were misaligned (Fig. IK), the rest being tangential, whilst all displaced myotubes in WT or cav-3 ^"Λ muscles were tangentially displaced. This was also true of the other stages examined (data not shown).The proportion of displaced myotubes declines with gestational age and is muscle group dependent (Fig. II). At El 3.5, 12% of myotubes in mdx intercostal muscles were incorrectly aligned, this declines with gestational stage but we found misalignment and tangential displacement of myotubes in mdx intercostals muscles at all gestational stages (E13.5-E17.5, Fig. II, M). At the same stages WT and cav-3^''^' intercostal myotubes are correctly aligned to the median and there are no deviating myotubes in WT or cav-3 -/- intercostals, suggesting the alignment mechanism in these muscles is tightly regulated and severely disrupted in mdx (Fig. H-N).

Morphological defects in dystrophic embryonic heart

Cardiomyopathy is a significant clinical consequence of both LGMD (1C) and DMD. We established the morphology and myosin localisation pattern of dystrophic and WT embryonic hearts using MF20 and cardiac β-myosin specific antibody N2.261 (Fig. 2). Consistent with the literature, skeletal fast myosin isoforms (FMyHC) could not be detected in embryonic hearts between E13.5 and E17.5 (My32 antibody, data not shown). Cardiac β-myosin is present in WT and dystrophic ventricular and atrial myocytes between E13.5 and E17.5 (Fig. 2A-F, I-P). Both mutants exhibit ventricular wall thickening (Fig. 2A-F) and cardiac myocyte disorganisation which is substantially worse in cav-3 ^" where myocytes form in a crisscrossed fashion overlapping one another (Fig. 2E-F and G-H). Atrial trabecular formation is also impaired in dystrophic embryos which in E14.5 cav-3^'1' embryos are short and stubby in contrast to mdx trabeculae which are long and hook shaped (Fig. 2I-J). At E17.5 there is distension or separation of the myocardial and endocardial cell layers of dystrophic atria and loss of cardiac β-myosin (Fig. 2K-P) in contrast to WT atria where staining is uniform and the two layers tightly adjoined (Fig. 2 K, N). There are distinct differences between the two dystrophic hearts; in cav-3^'1' at E17.5 there is patchiness and reduction in staining in the myocardial wall which is most evident in trabeculae (Fig. 20). In mdx, loss of staining is extensive in intertrabecular regions of the myocardium but is largely retained in the tips of trabeculae (Fig. 2P).

Myonuclei misplacement and fusion abnormalities

In E15.5 WT embryos, myonuclei are evenly spaced along the length of the myotube and, except in newly formed myotubes, are arranged evenly and helically around the edge (Fig. 3A, white stars). In mdx, myonuclei are more frequently centrally located and slightly further apart than WT (Fig. 3B). In contrast, cav-3^'A myonuclei are closer together and exhibit severely disrupted myonuclei spacing and nuclei 'bunching' which is particularly evident at the ends of myotubes (Fig. 3C-D). Transverse sections demonstrate the presence of peripheral and central nuclei at this stage (El 5.5) in WT muscle and show that in mdx and cav*^' muscles only central nuclei are present (Fig. 3E-G). To quantify these phenotypes we scored total myonuclei and myolϊbres over a fixed area, to establish that cav-3^'A myotubes contained twice as many myonuclei as those of mdx or WT, this is statistically significant at E 13.5, E15.5 and E17.5 (Fig. 3K; p< 0.05). There is also a substantial excess of myotubes in cav-3^v~ at these stages (p<0.05) and a smaller but significant (p<0.05) reduction in myotube number in mdx (Fig. 3H-J, L). The disproportion in myotube numbers in cav-3^~'^' and mdx embryos lessens between E 13.5 and El 7.5, but is statistically different to WT at all stages.

Hyperproliferation and apoptosis in cultured embryonic muscle stem cells

To characterise the embryonic muscle stem cell population we used dystrophic and wild type (El 1.5-E17.5) embryonic myoblast cultures (Smith and Merrick, 2008). The somite origin of these cells was established using myf-5 staining (Fig. 4). mdx and cav-3^'1' explants both have outgrowth rates which deviate from WT (Fig. 4A-B). Compared to WT, outgrowth of myf-5+ cells is significantly greater at all stages (El 1.5 - E17.5) in mdx and from E15.5-E17.5 in cav- 3^'1' . In earlier stages (El 1.5 - E13.5) cav-3^^' outgrowth rate is indistinguishable from WT suggesting defects occur later in this mutant. Myoblast proliferation (ki67 immunreactivity) and apoptosis (Dapi staining) were both significantly elevated in mdx between E11.5-E17.5 whilst in cav-3^'A apoptosis and proliferation rates equal those of WT until El 5.5 when there is a sharp elevation of both parameters to levels approaching those of mdx embryos (Fig. 4C, E). In mdx, proliferation (and apoptosis) decline from E13.3-E17.5, although they remain significantly higher than WT levels, the proliferation and apoptotic rates of cav-3^''^' myoblasts however, increase to E15.5 (Fig. 4C-F). At E17.5 the proliferative rate of cav-3^''^' cultures declines slightly, in line with mdx levels whilst apoptotic rate is maintained at the level of El 5.5 cav-3^''^" myoblasts (Fig. 4E). mdx explant derived soluble factors increase outgrowth of El 1.5 WT explants (Fig. 41).

The pax-7 skeletal muscle stem cell population is attenuated and disorganised in dystrophic embryos

At E17.5 the WT pax-7⁺ stem cell population is more sparsely interspersed between skeletal muscle myotubes although pax-7 protein continues to increase with muscle size ((Merrick et al., 2007); Fig. 5A-B, G-H, M & O). In WT embryos, pax-7 staining intensity is uniform and constant with gestational age (Fig. 5A-B, G-H & Q; lower proximal limb). Between E15.5 and El 7.5 both cav-3^''^' and mdx undergo attrition of their pax-7⁺ cell population throughout their musculature so that by El 7.5 there is significant reduction of pax-7 staining and pax-7 protein in both mutants (Fig. 5C-L, M, P; shown for lower proximal limb). This is particularly evident in cav-3^"'^' proximal hind limb muscles where pax-7 positive cells are almost absent at E 17.5 and those remaining stain very weakly (Fig. 5 J, white arrow). At El 5.5 {cav-3^'1' and mdx) and El 7.5 (mdx), pax-7 positive cell fragments are found in dystrophic muscles (Fig. 5L, indicated by a black arrow) suggesting these cells may be undergoing apoptosis. Mutant embryos (mdxcav-⁺'^~) which were deficient in dystrophin (mdx) and heterozygous for caveolin-3 (cav-3⁺'^~) have a very much more severe phenotype in the proximal limb where pax-7 positive cells are very sparsely present at El 5.5 (Fig. 5MN & R) and almost entirely absent at El 7.5 (Fig. 5O-P) in the lower proximal limb and very much attenuated throughout the musculature (see Figure 7).

Whole embryo immunoblotting demonstrates an increase in caveolin-3 protein in WT (and mdx) embryos at E15.5-E17.5 compared to El 1.5 andl3.5 and confirms its absence in cav-3^"'^' (Fig. 5M-N). In mdx caveolin-3 is not detected at El 1.5 (Fig. 5MN) but there is several fold increase of caveolin-3 content over WT at E13.5-E17.5 as measured by densitometry on 4 separate gel runs (p = 0.05 for all three stages; Fig. 50). Pax-7 content increases in WT embryos with gestation but is substantially reduced in cav-3^''^' and mdx at E15.5-E17.5 with the reduction being greater in cav-3^''^' than mdx at both stages suggesting caveolin-3 may regulate pax-7⁺ myoblast survival in late gestation. At El 5.5 there is reduction in the intensity of pax-7 bands of 15% and 60% respectively in mdx and cav-3^''^" dystrophic embryos (Fig. 5N, P). At E13.5 pax-7 is slightly elevated in cav-3^''^' and reduced in mdx , this may relate to the increase and decrease in myotube number found respectively in cav^^" and mdx embryos at this stage (see Fig.3) and suggests that developmental timing may be disrupted in these embryos.

Mis-localisation of fast myosin isoforms in dystrophic embryos follows a reciprocal pattern in caveolin-3 and dystrophin deficient embryonic muscles

In WT FMyHC is present in small numbers of secondary myotubes as early as El 1.5 and by El 5.5 is strongly localised to around 45% of secondary myotubes across a wide range of muscle groups. Around 80% of myotubes contain FMyHC at El 7.5 but staining intensity is heterogeneous and weak in many myotubes (Merrick et al., 2007). This dynamic and muscle specific pattern of fast-myosin localisation is disrupted in cav-3^''^' and mdx mutant embryos which show defects in developmental timing of FMyHC staining, in the total number of fast myosin positive myotubes present and in the intensity of fast myosin staining (Fig. 6, Fig. Sl). At E 13.5 FMyHC is elevated above WT in some cav-3^'1' muscles (notably the respiratory muscles) but substantially attenuated in all mdx muscles and in cav-3^''^' proximal muscles (Fig. 6A-F).

The appearance OfFMyHC⁺ myotubes is delayed in mdx embryos and in El 3.5 max embryos there are very small numbers OfFMyHC⁺ myotubes throughout the musculature, compared to WT or cav-3^''^' embryos at the same stage (respiratory muscles Fig. 6A-F; proximal muscles show a similar phenotype (not shown)). This finding is supported by immunoblotting where there is a statistically significant reduction in the intensity of the mdx E13.5 band (Fig. 6T & U) and by myotube counts (Fig. 6V) which demonstrate a statistically significant deficiency of FMyHC⁺ myotubes in E13.5 mdx embryos (p<0.05). A total of 6 mdx and 4 WT E13.5 embryos were used to establish these data. In max the staining intensity and proportion of FMyHC positive myotubes subsequently increases so that from El 5.5 -E 17.5 there is a substantial excess of My32 staining in dystrophin deficient myotubes compared to WT and increased staining intensity of FMyHC bands in whole embryo immunoblots (Fig. 6 G-O, T). Densitometry confirmed this conclusion revealing an increase in mdx and decrease in cav-3-/- embryo FMyHC content which are statistically significant at p<0.001 and p<0.05 respectively (Fig. 6U). Quantification of matched, FMyHC immunostained muscle sections in WT and dystrophic embryos established that the proportion of FMyHC positive myotubes is perturbed in cav-3^''^' and mdx embryos (Fig. 6V). Mdx embryos heterozygous for cav-3 {mdxcav-3*'^') also fail to downregulate FMyHC and have increased FMyHC staining throughout the musculature at E15.5 (Fig. 6R) and E17.5 (Fig. 6Q, S) even in muscles such as the intercostals where myotubes are severely depleted (Figure 7T-U).

Extensive intercostal muscle fibre loss and pax-7 depletion in mdxcav^+/~ mutant embryos

To establish whether up-regulated caveolin-3 was likely to be ameliorative or contributory to the mdxfDMD phenotype we generated double mutant embryos which were deficient in dystrophin (mdx) and heterozygous for the caveolin-3 null mutation (cav-3^+/~). At E15.5 these embryos have a 50% reduction of caveolin-3 compared to WT embryos (Fig. 5). Immunostaining with MF20 and pax-7 establishes that, at El 7.5, mdxcav-3^+/~ embryos have a more severe phenotype which in addition to loss of hind limb pax-7 myoblasts (see Figure 5) also includes a severe depletion of their intercostal muscle fibres which is accompanied by significant attenuation of pax-7 positive intercostal myoblasts (Fig. 7 A-J). The fibre-loss phenotype can be identified even at low magnification (Fig. 7A,D & F, I) by the presence of gaps ('white space') between the fibres of mdxcav^+/~ intercostals which in WT intercostal muscle fibres are densely packed together. At higher magnification (Fig. 7K-P) it is clear that this phenotype is a result of there being far fewer fibre clusters compared to WT, mdx or cav-3^+/~ intercostal muscles. This phenotype is very consistent, three different mdxcav^{+ ~} embryos are shown (Figure 7N-P; mdxcav-3^+/~\, 2 & 3). In mdx, intercostal fibres also appear more sparsely distributed than in WT suggesting a milder form of this phenotype. Quantitation of fibre density was achieved by counting intercostal fibre number over a fixed grid area and established that both mdx and het (mdxcav-3^+/') have reduced fibre densities, this is a statistically significantly reduction in the het mutant (p<0.05; Fig. 7R). When expressed as a proportion of WT it can be seen that mdxcav-3^+/~ mutant embryos have a massive 71.2 % depletion of their intercostal myotubes by E17.5 (Fig. 7S; mean of counts of three different El 7.5 mdxcav-3^+/~ embryos) whereas mdx have a depletion of one third (37.5%). A majority of these fibres express fast myosin (FMyHC) in both mutants Fig 7U.

Pax-7 staining of human muscle biopsy

This is shown as Figure 8 demonstrating that human juvenile muscles can be stained in a similar manner to that described above.

Pax-7 gene expression studies

RT-PCR was used to qualitise pax-7 mRNA levels. This showed lower pax-7 levels in DMD individuals than normal individuals. Igf-2 disrupted mice

Merrick et al (2007) describe Igf-2 in muscle fibres. Figure 9 shows Igf-2 in disrupted mice. Igf-2 message (rnRNA) is elevated in mdx and cow-3^"1" embryos. The suppression of Igf-2 in caveolin deficient embryos is at the post-transcriptional level.

Downstream genes of Igf-2

A decrease in CDKN1/P57kp2 or altered expression of pAkt (phosphorylated Akt) is indicative of muscular dystrophy, as shown by the expression levels in Figure 11. pAkt has a MD disease type specific response, for example in caveolin-3 deficiency/LGMD-lc pAkt is reduced compared to normal muscle and in mdx/DMD it is increased. The pattern of disturbance for pAkt is the same as that of Igf-2.

Discussion

In this study the inventors demonstrate the embryonic phenotype of mdx and cav-3^^' dystrophic mice and show that in mice mutant for both proteins {mdxcav-3^+/~) the embryonic phenotype is significantly more severe. We establish crucial roles for dystrophin and caveolin-3 in murine embyogenesis and establish that the pathologies which define the adult dystrophic phenotype and clinical picture of DMD and LGMD-Ic originate in failure of embryonic muscle differentiation. The key findings of this analysis are summarised in Table 1.

Early muscle patterning is disrupted in dystrophin deficient embryos

The growth behaviour of myf-5⁺ mdx embryonic myoblasts is disrupted from El 1.5 and mdx myf-5+ myoblasts are hyperproliferative and apoptotic. Myf-5 marks the embryonic myoblast population; it is expressed in myotome (E8.5) before myotube differentiation is initiated and is found in all muscle groups throughout myogenesis (Hadchouel et al., 2003; Ott et al., 1991). Dystrophin is expressed at a crucial stage (E9.5) in myotome differentiation, one day later than Myf-5 expression at E8.5, and 24 hours prior (E10.5) to the appearance of the first fully differentiated myotomal (epaxial) myotubes and the first expression of myosin heavy chain (MyHC) (Houzelstein et al., 1992; Ott et al., 1991; Schofield et al., 1993). Early hypaxial and secondary myogenesis are severely disrupted and delayed in mdx embryos as shown by the late appearance of FMyHC, pax-7 and cav-3 proteins and by the incomplete formation and disorganisation of mdx musculature between E11.5-E13.5 (Fig. 1; Fig. 6 and Fig. Sl in supplementary material). These data suggest dystrophin has an essential role in myotome differentiation which is disrupted in mdx embryos with severe consequences for patterning and function of the entire musculature.

Also at E10.5, myf-5+ myoblasts migrate from the myotome to initiate hypaxial muscle formation (Cusella-De Angelis et al., 1992). Myoblasts migrate under control of hox genes (pax3 and lbx) and differentiate in response to growth factors (wntl and shh) secreted by adjacent tissues, (Gross et al., 2000; Hadchouel et al., 2003). The cues which trigger embryonic myoblast migration are not known, in other tissues stem cell migration is regulated by guidance cues from originating and target tissues, is essential for correct patterning of embryonic structures and can be easily disrupted (Lehmann, 2001). It is probable therefore that E10.5-11.5 epaxial myotubes provide signalling cues which trigger migration of myf-5+ myoblasts from the myotome and initiate hypaxial and secondary myogenesis. In the absence of dystrophin these cues are absent and both the migration and initiation processes are impaired. The aberrant behaviour of El 1.5 WT explants cultured in El 1.5 mdxCM (Fig. 41) supports the view that El 1.5 myotome releases secreted factors which modify the behaviour of the El 1.5 embryonic myoblast population. Disruption of early myogenesis in mdx suggests these early role(s) for dystrophin are distinct from those of utrophin. This conclusion is consistent with published mRNA in situ patterns of dystrophin and utrophin which are exclusive in early embryonic stages (Houzelstein et al., 1992; Schofield et al., 1993). In later stages of gestation (see below) there appears to be a 'catch-up' process in mdx myogenesis which could be mediated by the subsequent over-production of caveolin-3 by these embryos or by compensatory expression of another protein, for example a-utrophin. In postnatal muscles a-utrophin is upregulated in dystrophin-deficient muscles and may take over some functions of dystrophin in mdx (Weir et al., 2004). The increased severity of the double mutant phenotype (mdxcav-3^+/~) in late gestation argues also for a compensatory effect of caveolin-3 in late gestation mdx embryos.

Caveolin-3 is regulated by muscle regulatory factors (MRF 's), activated during myotube differentiation and is expressed later than dystrophin in El 1.5 myotome (Biederer et al., 2000). This is consistent with our detection of caveolin-3 at El 1.5 in WT embryos. MRF's also regulate pax-7 which too emerges at El 1.5 (Merrick et al., 2007). In cav-3^''^' embryos the early muscle patterning process appears intact, myf-5+ myoblast behaviour is not disrupted and the localisation and timing of hypaxial muscle formation is comparable to WT. Caveolin- 3 does not therefore seem to be required for these early stages of myogenesis. In mdx, the appearance of both caveolin-3 and pax-7 is delayed, this could be as a consequence of the developmental delay in myogenesis in these embryos or more specifically downstream of the failure to activate dystrophin.

Hypaxial musculature and secondary myogenesis

Interpretation of the role of dystrophin in later embryonic events is complicated by the up- regulation of caveolin-3 in mdx embryos from El 3.5. In post-natal tissues, over-expression of caveolin-3 causes dystrophin down-regulation and a DMD-like phenotype (Galbiati et al., 2000). Loss of dystrophin causes breakdown of the DGC and suppression of dystrophin- associated proteins (Ohlendieck et al., 1993; Vaghy et al., 1998). However the muscles of DMD patients and adult mdx have 1.5 - 2.4 and 2-3 fold excess of caveolin-3 respectively (Vaghy et al., 1998). In E13.5-E17.5 mdx embryos we found a (stage dependent) average 3-5 fold excess of caveolin-3 suggesting that disruption of caveolin-3 expression is an early and significant consequence of dystrophin deficiency. Embryonic mRNA expression patterns of dystrophin, but not utrophin, overlap those of dystroglycan in WT and this pattern is unchanged in mdx embryos (Houzelstein et al., 1992; Schofield et al., 1995). Dystrophin may therefore negatively regulate caveolin-3 by competitive binding to β-dystroglycan (Ilsley et al., 2002).

In zebra fish embryos, caveolin-3 mutants have cytoskeletal and fusion defects and dystrophin mutants have unstable muscle attachments suggesting a role in placement and orientation of embryonic myotubes (Bassett et al., 2003; Nixon et al., 2005). E13.5 myogenesis is disrupted in cav-3^v~ and mdx, phenotypes suggest murine caveolin-3 and dystrophin have similar roles to zebra fish counterparts. In E 13.5 mdx, we found myotube displacement, splitting and branching. Myotube orientation defects are not found in caveolin- 3 deficient mutants. Instead embryonic cav-3^~A myonuclei are bunched to myotube ends suggesting the T-tubule defect reported post-natally for cav-3^^' (Minetti et al., 2002). Excess cay-3^'1' myotube production between E13.5-E17.5, 2-fold increase in myonuclei content and hypertrophy were also found. These data match those of an in vitro study using cultured cav- 3^"A myoblasts; which found a reciprocal phenotype (reduced myotube number, fewer myonuclei and hypertrophy) in caveolin-3 over-expressing myoblasts, suggesting caveolin-3 suppresses myoblast fusion (Volonte et al., 2003). E 13.5 mdx embryos partially reproduce the over-expression phenotype having hypotrophic myotubes and reduced myotube numbers. Myonuclei number is not affected. In later stages (E15.5-E17.5) however, although caveolin-3 levels remain elevated, mdx myotube numbers are comparable to WT. This suggests caveolin- 3 may regulate myotube size but the E 13.5 myotube deficit is due to delayed hypaxial myogenesis in mdx (see section above) rather than excess caveolin-3.

Reciprocal disruption of fast fibre specification in cav-3^^' and mdx

FMyHC+ myotubes appear first in WT epaxial muscles -El 1.5 and herald the start of secondary myogenesis (Merrick et al., 2007). Fibre-type switching begins at El 5.5 when some myotubes switch between slow and fast myosin expression and the process of establishing adult fibre-type ratios begins (Cho et al., 1994; Merrick et al., 2007). In mdx FMyHC+ myotube differentiation is significantly perturbed (see sections above) and fast fibre type specification is disrupted in both mdx and cav-3^'A (Fig. 6). Between E15.5-E17.5 there is a reciprocal phenotype where mdx has significant excess of FMyHC protein and a higher proportion of FMyHC+ myotubes compared to WT; in cav-3^~'^~ there is substantial reduction of FMyHC. The over-production of FMyHC could result from a "catch up process" in El 3.5 mdx muscles, facilitated by a compensatory increase in caveolin-3. However loss of FMYHC+ myotubes in El 5.5-17.5 cav-3^v' suggests caveolin-3 is also required for fast fibre- specification; that over-production of caveolin-3 in mdx is pathogenic for this phenotype and causes over-production of FMyHC fibres in E15.5-E17.5 mdx. Post-natal fast muscle fibres degenerate preferentially in DMD and mdx, with severe consequences for muscle function (Webster et al., 1988). In mutants heterozygous for caveolin-3 but entirely deficient for dystrophin (mdxcav-3^+/~), FMyHC+ fibres are still over-represented at El 7.5 compared to WT suggesting that it is the balanced relationship between dystrophin and caveolin-3 which is crucial for correct fast fibre proportion rather than caveolin-3 levels alone.

In adult tissues caveolin-3 and dystrophin interact directly by competitive binding to β- dystroglycan in the DGC (Sotgia et al., 2000). These data establish distinct roles for dystrophin and caveolin in myogenesis but suggest that both proteins and a functional DGC may be required for fibre-type specification. Loss of pax-7 myoblasts

E15.5-E17.5 cav-3^'1' and mdx exhibit pax-7⁺ myoblast attrition and significant depletion of pax-7 protein. Loss of pax-7 occurs rapidly in cav-3^~'^~ at El 5.5, a time-point when caveolin-3 is strongly up-regulated in WT embryos and the loss is greater than in El 5.5 mdx. Caveolin- 3 can elicit survival signalling in muscle as does pax-7 itself. Our data suggest a direct role for caveolin-3 in the rapid loss of pax-7+ myoblasts at E 15.5 in cav-3^'A and suggest that the attrition of pax-7+ myoblasts could be partially compensated in mdx muscles by their increased caveolin-3 levels (Relaix et al., 2006; Smythe et al., 2003). This conclusion is supported by the finding that in mdxcav^+/~ mutants pax-7 is depleted substantially more than in either single mutant such that by E 17.5 pax-7 cells are entirely absent in mdxcav^+A lower proximal limb muscles and are very much reduced in other muscles. PaX-T⁺ myoblasts are crucial for normal post-natal satellite cell emergence (Relaix et al., 2006; Seale et al., 2000). In the absence of pax-7 (pax-7^"7" mice) satellite cells are reduced in number and apoptosis is elevated. However, due to the presence of pax-3+ myoblasts, satellite cells are not entirely lost and there appear to be sufficient satellite cells to establish and sustain (post-natal) juvenile muscle development in these mice, however regeneration in adults is impaired (Oustanina et al., 2004; Relaix et al., 2006; Seale et al., 2000). The findings that E17.5 mdxcav^+/~ embryonic intercostal muscles are severely depleted of myotubes as well as of pax-7+ cells suggests that pax-7 may play an important role in embryonic myogenesis also. They suggest that dystrophic embryos have a reduced capacity for generating muscle fibres from late gestation which may be carried over to post-natal life. Dystrophic muscle regeneration is known to be abnormal being associated with myoblast apoptosis, hyperproliferation, irregular fibre size and progressive fibrotic deposition. Both mdx and cav- 3^~A muscles experience bouts of degeneration and regeneration in their muscles in the early post-natal weeks which are associated with elevated levels of apoptosis (Hagiwara et al., 2000; Smith et al., 1995). In mdx, there is progressive failure of muscle regeneration from 4 months, which, in mdx but not DMD, is ameliorated by concomitant reduction of degenerative processes which may, in part be mediated by up-regulation of utrophin (Reimann et al., 2000; Roig et al., 2004; Roma et al., 2004). Regeneration has not been studied in detail in adult cav-3^'A. The data suggest that the regenerative machinery of dystrophic mutants is abnormal at birth as these mutants are deficient in pax-7; this early loss could underlie the later progressive impairment of the regenerative process in adulthood. The significant loss of muscle fibre density in the respiratory muscles of E17.5 mdxcav^+/~ embryos together with a massive loss of pax-7+ cells further strengthens this conclusion and suggests that increased caveolin-3 levels play an important compensatory role in the degenerative phenotype of mdx (and potentially in the early stages of DMD).

Correlation between dystrophic mouse phenotypes and the clinical pathology of MD

DMD and LGMD Ic are early onset, progressive skeletal muscle diseases of children affecting cardiac and skeletal muscle function and muscle stability (Hoffman et al., 1987; Minetti et al., 2002). In DMD there is widespread, progressive mal-function of the entire musculature, abnormal caveolin-3 expression, myoblast apoptosis, cardiomyopathy and regeneration defects (Cox and Kunkel, 1997; Smith et al., 1995; Vaghy et al., 1998).

LGMD-IC exhibits similar but restricted myopathic changes particularly affecting the muscles of the limb, diaphragm and heart (Galbiati et al., 2001; Hagiwara et al., 2000; Smythe et al., 2003). mdx and cav-5^'1' post-natal phenotypes are sufficiently similar to the clinical pathologies of DMD and LGMD in most respects, to be widely used as disease models (Chamberlain et al., 2007; Chan et al., 2007; Galbiati et al., 2001; Hagiwara et al., 2000; Roig et al., 2004; Vaghy et al., 1998). The respective embryonic phenotypes of mdx and cav-3^'1' strengthen the validity of these mice as models for LGMD and DMD, provide new insight into the mechanisms underlying MD and the mode of function of dystrophin and caveolin-3 and suggest new approaches to dissecting the differences between the human and murine forms of the disease. The inventors identify key developmental stages; myotome differentiation (El 1.5) and secondary myogenesis (E13.5) at which dystrophin and caveolin- 3, respectively, play key roles and reveal two novel pathologies in late gestation; fast fibre specification and attrition of pax-7 myoblasts which provide insight into the mechanism underlying MD pathology and which suggest novel routes for therapeutic intervention and earlier diagnosis of MD.

Cardiomyopathy, particularly left ventricular failure is a significant clinical consequence of DMD and many other MD's and an established pathology of cav-3^'^, mdx and caveolin-3 over-expressing mice (Aravamudan et al., 2003; Cox and Kunkel, 1997; Hayashi et al., 2004; Quinlan et al., 2004; Woodman et al., 2002; Yue Y et al., 2003). Dystrophin and Caveolin-3 express in mouse embryonic heart at E9.5 and E10.5 respectively (Biederer et al., 2000; Houzelstein et al., 1992). At E13.5 mdx have moderate thickening of the ventricular apex and atrial trabecular defects. In cav-3^'1' there is severe thickening of the ventricle, atrial defects and a progressively worsening disruption of myocyte organisation characteristic of cardiomyopathy and consistent with the early onset post-natal cardiomyopathy seen in caveolin-3 deficient mice at 3-4 months. These data suggest caveolin-3 is required for normal heart development and has role(s) in cardiomyopathy, β-cardiac myosin is mislocalised in El 7.5 mdx and cav-3^^' and trabeculae are abnormal, β-cardiac myosin defects underlie some familiar cardiomyopathies and its disrupted expression in dystrophic hearts is therefore significant (Cuda et al., 1993). Atrial defects have not previously been reported for either mouse. The mdx finding suggests a developmental origin for a recent report of hypertrabeculation in a 28 year old DMD (Finsterer et al., 2005). Although post-natal mdx hearts are reported to have WT levels of β-cardiac myosin and increased levels of utrophin which may compensate for dystrophin deficiency these mice have a progressive cardiomyopathy (Quinlan et al., 2004). Localisation of β-cardiac myosin has not yet been established postnatally, its disrupted atrial localisation could therefore persist into the adult or may be lost in the peri-natal or juvenile period (Wilding et al., 2005).

Hyperproliferation and elevated muscle cell apoptosis are well-established, wide-spread features of post-natal MD muscle pathology which characterise both mouse and human forms of OMDI mdx and LGMD- lc/cav-3^'/' as well as most other MD types (Baghdiguian et al., 1999; Smith et al., 1995; Smith et al., 2000; Smythe et al., 2003). Dystrophin, dystroglycan and caveolin-3 have roles in survival signalling (Glass, 2005; Smythe et al., 2003). In mdx muscles, high levels of myoblast apoptosis and myoblast hyperproliferation are established from 1 week post-birth to adulthood (Smith, 1996; Smith et al., 1995; Spencer et al., 1997) and (in this study) during embryonic stages from El l.5 to El 7.5. These data suggest disrupted stem cell behaviour, and apoptosis are an early consequence of loss of dystrophin and an important contributor to the pathology of DMD. Myoblast apoptosis arises later in cav-3^'A at El 5.5 suggesting that this phenotype may be an accurate predictor of severity of disease.

New insights — significance for MD

These data offer a new perspective on the aetiology of muscular dystrophy, establish embryonic roles for dystrophin and caveolin-3 which progress understanding of myogenesis and suggest that pax-7 myoblast replacement and therapeutic strategies which seek to modify fibre-type specification might be productive in the early treatment of muscular dystrophy. Earlier diagnosis and therapeutic intervention are likely to improve the quality of life of muscular dystrophy patients. Moreover, the use of Caveolin-3, fast-myosin, Igf-2, CDKNl c/P57kip2 and pAkt in assays is also suggested.

Diagnostic assay further data

We have carried out pax-7 staining in human skeletal muscle samples as follows: control (undiseased) child, 31 days old (Figure 12 A-C); (un-diseased) human fetal tissues at 16 weeks (Fig. 12D-F) and 12.7 weeks (Fig. 12G-I). These data establish that the pattern of Pax- 7 staining in normal fetal and neonatal skeletal muscle is directly comparable (equivalent) to that found in mouse embryonic and neonatal tissues at equivalent stages. Pax-7+ myoblasts are abundant and evenly distributed in both human and mouse fetal and neonatal tissues and label strongly with pax-7 antibodies. We also examined pax-7 positive myoblast localisation in ten different (non-Muscular Dystrophy) muscle diseases, in each of these diseases pax-7 positive myoblasts were abundant and not significantly different in number to non-diseased muscle samples, a representative sample (myotubular myopathy) is shown in Figure 12 (J-L). In skeletal muscle samples from Duchenne and Becker's Muscular Dystrophy (DMD & BMD) patients however the result was different (Figure 12 M-P and figure 13). In DMD and BMD the numbers of pax-7 positive myoblasts were significantly depleted compared to controls and were consistent with disease severity (DMD muscles were consistently more depleted in Pax-7 cells than were those of the milder MD form, Becker's). The intensity of staining of Pax-7 was also reduced compared to normal samples and this was particularly evident in DMD patient muscles where Pax-7 positive myoblasts were few in number and Pax-7 predominantly weakly expressing. Large regions of DMD patient muscles contained no pax-7 positive myoblasts at all (Figure 12 M-P, weakly staining pax-7+ cells and regions with no Pax-7+ cells are indicated by a star *). These data demonstrate that pax-7 depletion is an early marker of DMD and BMD which can be used to identify these diseases from other non- MD myopathies and from non-diseased muscle biopsies in children. This marker can be used alone or in combination with a marker for muscle degradation (eg creatine Kinase), and/or for muscle regeneration (eg. developmental myosin, Igf-2) and/or other markers of the disease (eg caveolin-3, Fast-myosin) to identify Muscular Dystrophy in foetuses, neonates and young children prior to the onset of overt clinical symptoms (in DMD before birth, and from bith up to and including 5 years age.) These data also strongly support the use of Pax-7 alone, or in combination with other markers, as a marker of therapeutic efficacy and as an indicator of disease progression.

Further therapeutic data

Caveolin-3 is elevated above normal (wild-type) levels in mdx (mouse) embryonic and post natal tissues and in DMD (human) muscles. Double mutant mouse embryos which are deficient in dystrophin (mdx) and heterozygous for caveolin-3 have reduced levels of caveolin-3 and a more severe muscle pathology than that found in dystrophin deficiency (mdx) alone suggesting that elevated levels of Caveolin-3 may compensate for the loss of dystrophin. Figure 14 shows that when levels of caveolin-3 are reduced back to non-disease (wild-type) levels in mdx mouse that the post natal dystrophic phenotype of mdx also increases in severity confirming the therapeutic effect of increased levels of caveolin-3. In these mice (Figure 14). Using western blotting we have established that the levels of caveolin-3 protein found in these (DM - double mutant) animals is consistently reduced compared to those found in mdx mice alone and is equivalent to levels found in WT mice (Figure 14A). At the histological level the dystrophic pathology of mdx mice is most severe between 3-8 weeks post birth. Significantly the progression of the disease is prolonged in dystrophin deficient mice with WT levels of Caveolin-3 compared to mdx and there is a continued and progressive severe pathology in DM (het) muscles after 8 weeks (Figure 14B- C) compared to mdx muscle of the same age (9 weeks) or to the milder pathology seen in the caveolin-3-/- deficient mouse (cav-3-/-) (Figure 14D-E). Together these data establish that increased Caveolin-3 protein levels have a therapeutic effect on dystrophin deficient pathology and identify the upregulation of caveolin-3 as a valid treatment for Duchenne Muscular Dystrophy.

Igf-2 has an ameliorative effect on the dystrophic (mdx) phenotype (Smith et al, 2000) but the mechanism for this is unknown. Figure 15 demonstrates that Igf-2 can upregulate the expression of Pax-7, Pax-3 and caveolin-3 in dystrophic myoblasts suggesting a direct mechanism for its therapeutic effect and providing strong support for the use of this protein as a therapeutic agent separately or in conjunction with other treatments such as caveolin-3 (Figure 15).(A-B) Igf-2 induces increased expression (mRNA) of Pax-7 in WT and dystrophic myoblasts within 15 minutes of treatment of 10-20ug/ml Igf-2. The effect is prolonged and Pax-7 continues to be upregulated in myoblasts at least 24 hrs later (statistically significant pO.Ol, Studnet's t-test). (C-D) similarly Igf-2 induces upregulation of the Pax-3 message within 15 minutes and this is prolonged to at least 24 hours (20ug/ml Igf-2: p<0.01). (E-F) Dose reponse curve of Igf-2 treatment of Wt myoblasts showing that Igf-2 also induces an increase in pax-7 protein (shown here for 8 hours post treatment, p<0.01). (G) The ratio of caveolin-3 protein expression in WT and dystrophic myoblasts measured by western blotting illustrating that caveolin-3 is elevated in dystrophic myoblasts 2-3 fold above levels found in WT. Within 15 minutes Igf-2 treatment induces up to 8 fold increase in caveolin-3 protein in dystrophin myoblasts compared to WT and this is sustained to at least 24 hours (p<0.01, 20 ug/ml Igf-2).

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Claims

Claims

1. A method for identifying an individual exhibiting symptoms of, monitoring the treatment of or progression of, or having a propensity to develop muscular dystrophy comprising determining the level of expression in a tissue sample from the individual of one or more proteins selected from pax-7, caveolin-3 and/or fast-myosin.

2. A method according to claim 1 , wherein pax-7 is measured.

3. A method according to claim 1 or claim 2, wherein caveolin-3 is measured.

4. A method according to any preceding claim, wherein fast-myosin is measured.

5. A method according to any preceding claim, additionally comprising determining the level of expression of one or more of insulin-like growth factor-2 (Igf-2), CDKNlc/P57kip2 and/or pAkt, in the tissue sample.

6. A method according to any preceding claim, wherein the muscular dystrophy is Duchenne muscular dystrophy (DMD) and/or limb girdle muscular dystrophy (LGMD).

7. A method according to any preceding claim, wherein the tissue sample is from a baby before or after the birth of the baby.

8. A method according to any preceding claim, wherein the level of protein expression is determined by immunoassay.

9. A method according to claim 8, comprising determining the expression level of one or more of the proteins by

(i) immunostaining of the sample; and/or (ii) immunoblotting of proteins

10. A method according to any of claims 1 to 7, wherein the level of expression of each protein is determined quantitative polymerase chain reaction (QPCR).

11. A kit for use in a method according to any preceding claim, comprising two or more antibodies or pairs of PCR primers specific for, two or more of pax-7, caveolin-3 and/or fast myosin, or mRNA encoding such proteins.

12. A kit according to claim 11, wherein the protein is pax-7 protein or mRNA encoding pax-7.

13. A kit according to claim 11 or 12, wherein the protein is caveolin-3 or mRNA encoding caveolin-3.

14. A kit according to claims 11 to 13, wherein the protein is fast-myosin or mRNA encoding fast myosin.

15. A kit for use in a method according to claims 1 to 7, comprising an antibody specific for pax-7 protein, together with paraformaldehyde.

16. A kit according to claims 11 to 15, comprising an antibody or PCR primer pair specific for Igf-2.

17. A kit according to claims 11 to 16, comprising one or more reagents for tyramide signal amplification (TSA).

18. A method of treating a muscular dystrophy, comprising administering caveolin-3 to a patient or increasing caveolin-3 expression in a patient.

19. A pharmaceutical formulation, comprising caveolin-3 in combination with a pharmaceutically acceptable carrier.

20. Caveolin-3 for use to treat a muscular dystrophy.