JP2012525576A - Method for diagnosing and treating muscular dystrophy - Google Patents

Abstract

  The present invention relates to a method for identifying an individual exhibiting symptoms of muscular dystrophy or an individual having a tendency to develop muscular dystrophy, wherein pax-7, caveolin-3, and / or Alternatively, a method comprising determining the expression level of one or more proteins selected from fast muscle myosin is described. Also claimed is the use of caveolin-3 to treat muscular dystrophy and compositions containing the compounds.

Description

Assays The present invention relates to methods and assay kits for identifying individuals who have or are likely to develop muscular dystrophy such as Duchenne muscular dystrophy (DMD) and limb girdle muscular dystrophy (LGMD).

Duchenne muscular dystrophy (DMD) is the most severe family of debilitating congenital diseases associated with dysfunction of the transmembrane dystrophin-glycoprotein complex (DGC). Typical DMD affects the entire skeletal muscle tissue and is due to the loss of DGC, the essential intracellular component of dystrophin (Hoffman et al., 1987). Symptoms are characterized by progressive impairment of early (about 3 years) skeletal muscle function, leading to extensive muscle damage and causing premature death (usually in the 20s). However, complications that generally result from respiratory muscle dysfunction become death hospitals, with 95% of patients with DMD developing cardiomyopathy, of which 10-30% are the leading cause of death ( Cox and Kunkel, 1997). Mdx mice, a dystrophin-deficient model of DMD, exhibit many of the postnatal features of DMD, including cardiomyopathy; skeletal muscular dystrophy associated with the onset of fiber regeneration; fibrosis; skeletal myoblast hyperproliferation and apoptosis (Harper et al., 2002; Smith et al., 1995). The phenotype is less severe than human DMD, although mdx lifespan is reduced compared to wild type (Chamberlain et al., 2007). Loss of caveolin-3 results in a mild form of MD, LGMD-1c, where the affected muscle groups are primarily limb glands and heart, and caveolin-3 deficient mice (cav-3 ^{− / −} ) are LGMD,
Shows T-tube deficiency, cardiomyopathy, and skeletal muscle apoptosis (Hagiwara et al., 2000; Minetti et al., 2002).

Embryonic skeletal muscle is derived from the segmental cutaneous muscle plate region (Cossu et al., 1996; Hollway and Currie, 2003). In chicken embryos, all regions of the dermal sarcomere appear to provide myogenic precursors to this process in a time-dependent manner (Gros et al., 2004). Dystrophin is
It is first expressed in E9.5 derived embryonic somites and thus may have a function in early myogenesis (Huang et al., 2000; Ilsley et al., 2002). The musculoskeletal muscles (from E10.5) on-axis (deep back muscles) and (from E11.5) on-axis (limbs, abdomen, diaphragm) muscles, respectively, under the control of inducers secreted by surrounding tissues. Produces cells that will generate (myf-5 ⁺ ) stem cell populations (reviewed in Hollway and Currie, 2003).

  In mammalian embryonic skeletal muscle differentiation, it is believed that there are two myogenic surges that produce an off-axis lineage to produce a primary myotube scaffold, and then a number of secondary myotubes, which are nascent muscles. (Cossu et al., 1996). The secondary myotubes are a subset of morphologically distinct myotubes that are longer and thinner than the primary myotubes that form around a single larger primary myotube (Cho et al., 1994). .

Myotube differentiation depends on the presence of a functional muscle stem cell population. Myf-5 is expressed in somites and is the earliest myogenic regulator (MRF) in E8.5, the only MRF found before muscle differentiation, both primary and secondary myogenesis Persist in all embryonic muscle groups (Cossu et al., 1996). Therefore, Myf-5 is a good marker for embryonic myoblast population. Phylogenetic analysis suggests that the majority of satellite cells are derived from somites (Armand et al., 1983). With extensive evidence, now the adult satellite cell population
The pax-7 + cell population has been associated, particularly as essential to the correct functionality of its repair function (reviewed in Zammit et al., 2006). Pax-7 is a transcription factor and repressor of myogenesis, plays an important role in the maintenance and specialization of adult skeletal muscle stem cell population (“satellite” cells), and emerges from E11.5-derived somites. It is expressed in differentiated muscle stem cells (Merrick et al., 2007: Relaix et al., 2006; Seale et al., 2000).

  Specificity of individual skeletal muscle function depends on the proper localization of fast muscle myosin isoforms into different myotubes, a process initiated during late pregnancy (Merrick et al., 2007) . In mammalian embryos, developing (embryonic and neonatal) myosin isoforms are co-expressed in neonatal secondary myotubes with adult fast muscle myosin isoforms (Cho et al., 1994). In the late phase, the developing myosin isoform is down-regulated and replaced by an adult myosin heavy chain (MyHC) isoform in a muscle-specific pattern, and this process does not end until several weeks after birth (Agbulut et al., 2003; Merrick et al., 2007). The embryonic heart expresses two myosin isoforms (myocardial myosin alpha and slow / myocardial myosin beta) in a time-controlled manner. β-myocardial myosin mutations are involved in cardiomyopathy (Geisterfer-Lowrance et al., 1990).

Dystrophin is an essential component of DGC, a multifunctional protein complex that binds to the beta subunit of dystroglycan inside the cell membrane and binds the extracellular matrix to the actin cytoskeleton of skeletal muscle myotubes ( Ervasti and Cambell, 1993). In postnatal muscle, dystrophin deficiency results in complete disassembly of DGC and secondary downregulation of the majority of DGC proteins (Ohlendieck et al., 1993). Caveolin-3 is localized to both skeletal muscle caveolae and DGC and binds to the same PPXY motif at the β-dystroglycan c-terminus, which recognizes and binds dystrophin by a specific WW domain, and thus dystrophin and β-dystrophin. Blocks interaction with glycans (Jung et al., 1995; Sotgia et al., 2000).

Competitive interactions of caveolin-3 and dystrophin to the β-dystroglycan binding site may be important for several aspects of muscle development, but this has not been extensively studied. All three genes are expressed early in development in chicken embryos, mouse embryos, zebrafish embryos, and Xenopus 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 zebrafish, loss of either of these proteins has been shown to impair myogenesis and cause gross myopathy, and in mice, dystroglycan deficiency leads to early embryonic lethality (Bassett et al. 2003; Nixon et al., 2005; Parsons et al., 2002; Williamson et al., 1997).

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  We have investigated the impact of caveolin-3 and dystrophin loss on skeletal muscle development to demonstrate the functional role of these two proteins during myogenesis and to identify assays for early diagnosis of muscular dystrophy did.

By investigating two different but functionally related skeletal muscular dystrophy mutants (mdx and cav-3 ^{− / −} ), the key elements of pathology of DMD and LGMD1C types were found to be embryonic cardiac muscle and skeletal It was demonstrated for the first time that it was caused by an obstacle in the process of muscle pattern formation. Impaired myogenesis occurs earlier in mdx than in cav-3 ^{− / −} , with a milder LGMD-1c phenotype, consistent with early (E9.5) expression of dystrophin. Myogenesis was severely impaired in mdx embryos showing delayed development, myotube morphology and replacement defects, and abnormal stem cell behavior. Caveolin-3 protein is elevated in mdx embryos. cav-3 ^{− / −} (from E15.5) and mdx (from E11.5) are both hyperproliferation and apoptosis of myf-5 + embryonic myoblasts, depletion of pax-7 + myoblasts in situ, And depletion of total pax-7 protein at late pregnancy. Both have heart defects. cav-3 ^{− / −} has a more limited phenotype, including ventral muscle deficiency, excess, dysplastic hypertrophic myotube, doubling of myonuclei, and reduced fast myosin heavy chain (FMyHC) content Exists. Several mdx embryo pathologies, including myotube failure, myotube number reduction, and FMyHC increase, are reciprocal with cav-3 ^{− / −} . In dystrophin-deficient (mdx) and caveolin-3 heterozygous (cav-3 ^+/- ) double mutant (mdxcav ^+/- ) embryos, caveolin-3 is 50% of the wild type Reduced, these phenotypes are severely exacerbated, intercostal muscle fiber density is reduced by 71%, pax-7 positive cells are completely depleted from the lower limbs, markedly reduced elsewhere, and increased caveolin-3 levels Data suggesting a compensatory role rather than a contributing role was found in mdx embryos. These data demonstrate the important role of dystrophin in early muscle formation and demonstrate that caveolin-3 and dystrophin are essential for the creation of precise fiber type specialization and stem cell function. These data are very useful in bridging important gaps in the natural course of MD, establishing an initial diagnosis of DMD / LGMD, and designing initial treatment protocols that lead to better clinical outcomes for these patients.

  Traditional Duchenne muscular dystrophy assays involve searching for polymorphisms in the dystrophin gene. This gene is large and not all polymorphisms are tested. For example, testing for 19 mutations identified only 65% of cases. Currently, this test is performed only if there is a family history of the disease. However, approximately one third of all cases are novelty that occurs spontaneously in individuals.

  The tests identified by the present inventors are not based on polymorphisms in the dystrophin gene and are potentially applicable to all forms of muscular dystrophy. Furthermore, this test can be applied to both prenatal and postnatal infants. More conventional forms of diagnosis are not performed until the child reaches 3-5 years of age. This assay allows early diagnosis of the disease, thereby allowing early treatment of the disease (eg, by replacing pax-7 positive stem cells).

  Pax-7 / Myf5 cell stem cells have been suggested for use in therapeutic applications (WO 2007/059612). Such cells have been suggested as myogenic progenitor cells for use in the treatment of muscle diseases including muscular dystrophy. These cells are used to treat this disease as a source of new muscle cells. However, it has not been suggested to use Pax-7 as a marker for muscular dystrophy. This protein is simply used as one of two markers to identify the subpopulation of stem cells used.

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

  The method can be used to identify individuals who are exhibiting symptoms of the disease or individuals who have a tendency to develop symptoms.

  If treatment or progression is monitored, the level in the sample can be compared to the level in a sample from a previously collected individual, such as days, weeks, or months ago.

  The expression level of this or each protein may be compared, for example, to a predetermined level associated with an individual who does not show symptoms of muscular dystrophy or does not have a tendency to develop muscular dystrophy, and / or muscular dystrophy May be compared to a predetermined level previously identified as being associated with an individual exhibiting or having a tendency to develop.

  Pax-7 and caveolin-3 have been identified as particularly relevant for the initial determination of whether, for example, infants or preterm infants are likely to develop 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 with or without measuring fast muscle myosin.

  These two markers (pax-7 and caveolin-3) make it possible in particular to identify early the likelihood that an individual will develop muscular dystrophy. Until now, it is necessary to wait for the onset of the disease, which is often not recognized until the individual is several years old. Alternatively, for example, there are numerous prior art assays that look for specific polymorphisms in the dystrophin gene. However, such assays are typically directed to polymorphisms associated with certain families with a family history of such polymorphisms. Furthermore, the accuracy of such assays is typically still low.

  Thus, the tissue sample may be collected from the individual before the individual's birth or may be collected after the birth. Myogenesis typically occurs from 8-9 weeks of gestation. Therefore, potentially, samples after that period may be collected. The individual may be, for example, a child, eg, less than 16 years old, less than 12 years old, less than 10 years old, less than 6 years old, less than 3 years old, less than 2 years old, or less than 1 year old, and may still be in utero. Individual patients may be between 3 and 6 months of age from birth.

  Muscular dystrophy (MD) refers to a group of genetically inherited muscle diseases that weaken the muscles of mammals such as humans. MD is usually characterized by progressive skeletal muscle weakness, loss of muscle proteins, and death of muscle cells and tissues. Preferred diseases include Duchenne (DMD), Limb-girdle (LGMD), Emerydrefus, Ophthalopharyngeal, Distal, Myotonic, Congenital, Becker, and / or Facial Scapulohumeral It is. The disease may be DMD and / or LGMD.

  Typically, decreased expression of pax-7 or altered expression of caveolin-3 compared to a normal individual indicates an individual who is showing or has a tendency to develop symptoms of muscular dystrophy.

  The individual may be a human individual, but there are many different animals that develop muscular dystrophy-like symptoms. Such animals are typically mammals and include mice, rats, sheep, dogs, horses, cats, and non-human primates.

  Fast muscle myosin may be measured. The inventors have determined that fast muscle myosin exhibits different expression levels in different diseases. For example, fast muscle myosin is increased in a mouse model of Duchenne muscular dystrophy but decreased in limb girdle muscular dystrophy. Thus, assaying the expression level of the protein would indicate a high probability of developing this disease. Preferably, fast muscle myosin is assayed in combination with one or both assays of pax-7 and / or caveolin-3. Fast muscle myosin can also be used alone to diagnose certain types of muscular dystrophy such as, for example, DMD and LGMD.

  The assay may further comprise determining the expression level of insulin-like growth factor-2 (Igf-2) in the tissue sample. Igf-2 is overexpressed in muscular dystrophy such as Duchenne muscular dystrophy, and is reduced in caveolin-3 deficient LGMD. This also provides a prognostic indicator of the disease.

  In addition, the following gene products downstream of Igf-2 may be assayed: CDKN1c / P57kip2 and / or pAkt.

Igf-2, CDKN1c / P57kip2, and / or pAkt can also be assayed separately from other proteins. Thus, for example, Igf-2 may be assayed separately without measuring pax-7 and caveolin-3. Igf-2 may be used with CDKN1c / 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 a growth factor expressed in the fetal period. . Although this was known, it was not known that it was specifically expressed in skeletal muscle myotubes. The main finding of this paper was that Igf-2 affects the proportion of fast muscle myosin positive fibers.

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

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

  Protein expression can be determined by measuring protein expression levels either directly or indirectly by determining mRNA levels in the sample.

  Typically, the level of protein expression can be determined by immunoassay. Antibodies specific for the above proteins are generally known in the art. Such antibodies can be labeled directly or indirectly by methods generally known in the art. For example, a tissue sample can be immunostained with an appropriately labeled antibody. The sample can be fixed before staining. The inventors have found that paraformaldehyde is particularly effective when used with antibodies specific for proteins, particularly antibodies specific for pax-7. Preferably, therefore, the fixative used is paraformaldehyde. The binding between the protein and the antibody can be determined by methods generally known in the art. For example, the avidin-biotin method is generally known in the art. In such assays, horseradish peroxidase is typically used in combination with a substrate such as 3 ′, 3, 5, 5 ′ tetramethyl-benzidine to form a colored product that is then used, for example, under a microscope. Can be visualized. The inventors have found that tyramide signal amplification (TSA), particularly when used with pax-7, results in particularly good staining. Therefore, preferably TSA is used for immunostaining.

  Alkaline phosphatase can also be used. For this, a preformed cyclic enzyme anti-enzyme immune complex composed of three enzyme molecules (alkaline phosphatase) and two antibody molecules is used. This technique can typically be visualized by using a blue dye (Fast Blue BN) or a red dye (Fast Red TR).

  Immunoassays can be visualized using directly labeled anti-protein antibodies. Alternatively, they may be visualized indirectly by using anti-immunoglobulin antibodies that bind to anti-protein antibodies. The secondary antibody may itself be labeled.

This antibody or each antibody may be labeled with a phosphor. Such phosphors are generally known in the art. They include, for example, fluorescein, fluorescein isothiocyanate (FITC), and rhodamine. Such phosphors emit different colors of fluorescence. Preferably, therefore, the antibodies used in this assay (and indeed the kits defined below) have different fluorophores and / or have different visualization methods, for example using horseradish peroxidase , Allowing various proteins to be assayed from the same tissue sample.

  Proteins can be visualized by adding labeled antibody to a tissue sample, partially purified sample, or purified protein obtained from the sample to identify total antibodies bound to the sample. The protein in the sample may be purified and visualized by immunoblotting.

  Alternatively or additionally, protein expression levels may be determined by identifying the mRNA levels expressed in the tissue sample. The level of mRNA expression may be determined by quantitative polymerase chain reaction (QPCR) or real-time PCR (RT-PCR). In this technique, a pair of primers specific for the mRNA encoding the protein is used. Such techniques are generally well known in the art.

  There are many commercial systems in the art that allow quantitative PCR using, for example, SYBR ™ staining. Manufacturers include Applied Biosystems Limited and Promega Corporation.

  The invention relates to two or more antibodies specific for two or more of pax-7, caveolin-3 and / or fast muscle myosin, or two of pax-7, caveolin-3 and / or fast muscle myosin. Also provided is a kit for use in the method according to the invention comprising a pair of PCR primers specific for the mRNA encoding the above.

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

  In addition, the protein may be fast muscle myosin or mRNA encoding fast muscle myosin.

  The antibody may be directly labeled with a suitable label, such as one of those described above. Such labels include fluorescent labels and labels based on enzymes such as alkaline phosphatase or horseradish peroxidase.

  The kit may include an antibody against one protein and a pair of primers for a second protein. Alternatively, the kit may contain PCR primers for two or more other proteins. The kit may contain two or more antibodies against different proteins. In the latter case, the antibody may be labeled with a different label to allow a substantially simultaneous measurement of the amount of each protein.

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

  The kit may further comprise an antibody specific for Igf-2 or a PCR primer pair.

Where antibodies are provided in the kit, the kit may include one or more reagents for tyramide signal amplification. This may include, for example, biotinylated tyramide.

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

  The kit may include instructions for use with the kit. The instructions may include details regarding, for example, typical levels of protein expression associated with individuals who are showing symptoms of muscular dystrophy or who have a propensity to develop muscular dystrophy. The instructions may include details regarding expression levels in normal individuals. This allows comparison of data obtained from samples derived from the compared individuals with a predetermined value in order to allow the practitioner to evaluate the validity of the acquired data. One or more controls with known concentrations of protein may be provided.

  Where PCR primers are provided, the kit may further include one or more labels or other reagents to allow for performing quantitative PCR. This may include, for example, SYBR ™ green.

  The kit may also contain a predetermined concentration of one or more protein control samples for use as standards in the assay.

  The results obtained by the inventors also show that caveolin-3 can be used to relieve the symptoms of muscular dystrophy.

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

  Utrophin has been demonstrated to be useful in the treatment of muscular dystrophy. For example, U.S. Patent Application No. 2009/054327, U.S. Patent Application No. 2008/160108, and WO 97122696, which are hereby incorporated by reference in their entirety, include such proteins as such treatments. The method used in is disclosed.

  Also provided is a pharmaceutical formulation comprising caveolin-3 in combination with a pharmaceutically acceptable carrier. The present invention also provides caveolin-3 for use in treating muscular dystrophy.

  Hereinafter, the present invention will be described with reference to the following drawings by way of example only.

(A) Skeletal myotube branching of mdx myotubes: (IC) intercostal space, (L) limb, (F) facial muscle group; 0: no abnormality detected. (B) Myotube branching, E15.5 mdx biceps. Inset, section enlargement. Staining: hematoxylin and eosin. (C-H) Pan-myosin (MF20), proximal muscle, blue bars indicate wild-type (WT) myotube width (C, F) WT; (D, G) mdx, stunted myotubes, black arrows are Myotube division; (E, H) cav-3 ^{− / −} , hypertrophic myotubes. (FH) (F) Various myotube diameters of (G) mdx and (H) cav-3 ^{− / −} muscle fibers compared to (WT) WT. (I) Misplaced myotubes as a percentage of total count (mix of tangential and misaligned myotubes). 0: No misplaced fibers (J) Scoring strategy for misplaced myotubes:> 25 ° from the midline is tangential (T), <25 ° is misaligned (M). (K) Proportion of misaligned mdx myotubes at E13.5. (L to N) E13.5 IC muscle, MF20; (M) mdx misaligned myotubes (black arrows, M) (L) WT (N) cav-3 ^{− / −} has no myotube misplacement. Error bar: s. d. Size bar 20 micrometers. Student t-test; ^* p <0.01; ^** p <0.001, comparison with WT value. (A-F, I-N) Central section heart immunostained for β-myocardial myosin heavy chain (N2.261) E13.5 (A) WT; (B) mdx; (C) (D) Compared with WT Cav-3 ^{− / −} , E14.5 ventricular apex structure showing mild and (C) extensive ventricular wall hypertrophy, tissue collapse in cav-3 ^{− / −} (E), and mdx (F ) Is not so good. (GH) MF20 stains E15.5 cav-3 ^{− / −} heart, (G) Tissue collapse of ventricular wall, (H) cardiomyocytes cross each other. The atrial trabeculae (tb) of (I to J) E14.5 is short and thick in (I) cav-3 ^{− / −} and is hooked in (J) mdx. (K-P) E17.5 atrium; (L-P) (K, N) (L, O) cav-3 ^{− / −} trabecular (tb) and (M, P) mdx atrium compared to WT Reduction of N2.261 label and cell layer spreading in the wall. Size bar 20 micrometers. (Q) Low magnification image of MF20 labeled mdx heart to illustrate heart orientation and coincidence sectioned sagittally through the most central portion of the heart. E15.5 Proximal myocardial positioning: (A) WT, spiral, uniform spacing; (B) mdx, central position; (C) cav-3 ^{− / −} , near central position, non-interval Rules. A white star ( ^* ) shown overlaid, one myotube nucleus; small white arrow, myonuclei. In the wild type (A), a small white arrow indicates the peripheral nucleus of two separate myotubes. The peripheral nuclei are not evident in mdx and cav-3 ^{− / −} muscles (BC). (D) Clustering of myonuclei at the end of myotubes, cav-3 ^{− / −} . (EG) E15.5 WT (E), mdx (F), and (G) cav-3 ^{− / −} the cross section of the myotube of the low proximal limb is the peripheral myonuclei (arrows), wild type But is not associated with dystrophic embryo myotubes at this stage. mdx stunting and cav-3 ^{− / −} myotube hypertrophy can also be observed; the green line indicates wild type myotube diameter. Low magnification images of (H) WT, (I) mdx, and (J) cav-3 ^{− / − in} the low proximal limb region show a decrease in mdx fiber density compared to wild-type matched embryo sections. And cav-3 ^{− / −} muscle fiber density increase. (K) The number of myonuclei is doubled in cav-3 ^{− / −} (E13.5 to E17.5) compared to the wild type; ^* p <0.05. Slight (not statistically significant) reduction of mdx myonuclei (E13.5-E17.5). The average myocardial content of myotubes is constant at E13.5-17.5. (L) Increase in cav-3 ^{− / −} (E13.5-E17.5) myotube density and decrease in mdx (E13.5) myotube density compared to wild type. For each strain and stage, myotubes were counted in a constant matched muscle area, ^* p <0.05. Size bar 20 micrometers. (A) The outgrowth rate of embryonic myoblasts derived from muscle explant cultures is mdx derived from E11.5 and E15.5 compared to wild type explants cultured at the same time. Increased at cav-3 ^{− / −} of ˜E17.5. (B) Myf-5 immunostained explant. (C) Hyperproliferation of embryonic myoblasts (D) in mdx from E11.5 and cav-3 ^{− / −} from E15.5 as determined by Ki67 + immunoreactivity. (GH) E15.5 primary cultured wild-type embryonic myoblasts (G) Myf-5 staining (H) secondary antibody control. (I) The outgrowth rate of E11.5 wild type explants is increased in E11.5 mdx explant conditioned medium (mdx CM) (p <0.05, ^* ), but cav-3 ^{− / -} In or WT CM do not proliferate. Error bar, s. d. . (E) Increased apoptosis from E11.5 with mdx and E15.5 with cav-3 ^{− / −} (F) Dapi staining, arrows indicate apoptotic cells. ^* P <0.05, from WT; ^** p <0.01, from WT; ^# p <0.05, mdx and cav-3 ^- / - between. Pax 7 immunostaining in WT, mdx, cav-3 ^{− / −} , and mdxcav ^+/− lower proximal limbs; (AF, MN, QR) E15.5 (GL) E17.5. (A to E; G to K) Low magnification; (B to F; H to L) High magnification; Size bar 10 micrometers. Q-R WT (Q) and mdxcav-3 ^+/- Global field of view of the low proximal limb. mdxcav ^+/- , cav-3 ^-/- , and mdx putative apoptotic (fragmented) pax-7 ⁺ nuclei, E15.5 WT (B, D, and F) and (L) E17.5 mdx (black arrow) is not apoptotic. Pax-7 staining decreased, cav-3 ^{− / −} and mdxcav-3 ^+/− white arrows (CD and MN) E15.5; (I to J) E17.5. (M to R). At E17.5, the mdxcav-3 ^+/− low proximal limb muscle lacks pax-7 + cells (OP). (S) Immunoblot, (i-iii) Caveolin-3, (iv) pax-7. Contact (i) 10 seconds; (ii) 1 minute; (iii) 4 minutes. (Iv) Pax-7 protein is reduced in E15.5 and E17.5 dystrophic embryos. With a 4-minute contact, pax-7 is detected only with an E11.5 WT. (T) Densitometric analysis, caveolin-3: α-tubulin ratio confirms an increase in caveolin-3 in mdx. ^* p <0.05; The (U) table shows an estimate of the fold increase in caveolin-3 in mdx (compared to the wild type) after analyzing the three embryonic stages separately (4 experiments per stage). P = 0.05 for all periods. (V) Densitometric analysis, pax-7: α-tubulin demonstrates the reduction of pax-7 protein in cav-3 ^{− / −} and mdx (E15.5 to E17.5). E13.5 cav-3 ^{− / −} embryos contain significantly more pax-7 than wild type. ^* p <0.05; (T, V) Mean ± s. of two separate experiments. d. . (W) Immunoblots show E15.5 mdx embryo brothers in which caveolin-3 protein is reduced in E15.5 mdxcav-3 ^+/− embryos (50% relative to WT ^# ) and caveolin-3 is increased. It is demonstrated that it is significantly reduced compared to Neomycin immunostaining confirms the presence of the cav-3KO transgene in cav-3 ^{− / −} and mdxcav-3 ^+/− . The reduction in neomycin staining seen in mdxcav-3 ^+/− embryos compared to cav-3 ^{− / −} reflects that they are heterozygous for the cav-3KO transgene. # Densitometry: caveolin-3: α-tubulin ratio, track 1 WT compared to tracks 3 and 4, mdxcav-3 ^+/- . (A to F) FMyHC immunostaining with E13.5, (A to C) Diaphragm, (D to F) Intercostal space. (A, D) WT, (B, E) mdx, and (C, F) cav-3 ^{− / −} are the reduction of FMyHC in (B, E) mdx and (C, F) cav-3 ^{− / −. -} increase of FMyHC in the respiratory muscles. (G to L) FMyHC (G to I) diaphragm in E15.5 respiratory muscles; (J to L) intercostal (G, J) WT; (H, K) mdx (I, L) cav-3 ^{− / −} . (M) Increased FMyHC staining in (N) mdx and (O) decreased FMyHC staining in cav-3 ^{− / −} compared to WT E17.5 proximal limb. In (PS) mdxcav-3 ^+/- , FMyHC is E17.5 (Q) and (S) (proximal limb shown) and E15.5 (R) (intercostal and diaphragm are shown) It is also increased compared to the wild type. Comparison of cav-3 ^{− / −} (P) and (Q) mdxcav-3 ^+/− of E17.5 showed that dystrophin deficiency was down-regulated in FmyHC seen in cav-3 ^{− / −} and wild type embryos It is demonstrated to suppress An excessive loss of FMyHC in cav-3 ^{− / −} at E17.5 compared to the wild type embryo (M) can also be clearly seen (Q). (T) Wild type, cav-3 ^{− / −} , and mdx immunoblots show a decrease in FMyHC in cav-3 ^{− / −} and delayed production of FMyHC in mdx embryos compared to wild type. (U) Densitometry, FMyHC: α-tubulin ratio demonstrates statistically significant increases and decreases in FMyHC content in E17.5 mdx and cav-3 ^{− / −} embryos, respectively. At E13.5, the FmyHC of cav-3 ^{− / −} embryos is significantly excessive; the mean ± sd of two separate experiments. d. . (V) FMyHC positive myotubes expressed as a percentage of the total count in wild type embryos, mdx embryos, and cav-3 ^{− / −} embryos (E13.5, E15.5, and E17.5). Error bar, s. d. . ^* p <0.05; ^** p <0.01. Size bar 20 micrometers. Panmyosin (MF20) (B, E, G, J) or pax-7 (A) to show extensive loss of pax-7 myoblasts and muscle fiber density in muscle of late pregnancy (E17.5) embryo , CD and F, HI) (AE) wild type and (FJ) cdx-3 mice (mdxcav-3 ^+/− ) heterozygous for caveolin-3. Higher magnification images of (K-P) MF20 labeled intercostal muscle sections (corresponding to the 4th intercostal space of each embryo) are (K) WT, (L) mdx, (M) cav-3 ^{− / −} , and ( (NP) shows differences in myotube density among three different mdxcav-3 ^+/− E17.5 embryos. E17.5 WT, mdx, and mdxcav ^+/- (het) Fiber density evaluated in (R) constant grid area of embryonic intercostal muscle, and (S) Percent reduction in fiber density. The E17.5 intercostal range of (T) WT and (U) mdxcav ^+/− labeled with My32 antibody indicates that almost all fibers are FMyHC positive. Note that fibers downregulate FMyHC in many WTs, but not mdxcav-3 ^+/− intercostal fibers. Pax-7 human muscle biopsy: Pax-7 immunostaining is performed to identify pax-7 + skeletal muscle stem cells of stored human juvenile muscle biopsy. Pax-7 labels the cell nucleus. Dark staining indicates pax-7 + nuclei. Light staining is a hematoxylin counterstaining that indicates the location of pax-7 negative cell nuclei. RT-PCR analysis (A) Densitometric analysis of pax-7 mRNA expression in 6 WT and 6 mdx mouse myoblast isolates statistically significantly suppressed Pax-7 expression in dystrophic myoblasts It is demonstrated that Data shown are the mean and standard deviation of the densitometric results of individual myoblast isolates. (B) RT-PCR shows detection of pax- & mRNA in 6 WT and 6 mdx myoblasts. Pax-7 expression was consistently suppressed in dystrophic (mdx) myoblasts compared to WT. Igf-2 immunostained embryonic skeletal muscle damaged in MD mice for insulin-like growth factor 2 (Igf-2) from E14.5 to E17.5. (AD) Wild type (WT) muscle shows a characteristic striped pattern of Igf-2 and 50% of the fibers are positive for Igf-2 immunostaining. (EH) Dystrophin deficient (mdx) muscle overexpresses Igf-2 from E14.5 to E17.5 and loses the WT “striped” immunostaining pattern. In contrast, (IL) Igf-2 is lost in caveolin-3 deficient (cav-3 ^{− / −} ) muscle. (M) Quantification of the immunostaining pattern confirms these conclusions. (N) RT-PCR demonstrates that Igf-2 message (mRNA) is elevated in mdx and cav-3 ^{− / −} embryos. Thus, suppression of Igf-2 in caveolin-deficient embryos is at the post-transcriptional level. Downstream genes of Igf2 Downstream genes of Igf-2 (CDKN1c / P57kip2 and pAkt) are also damaged in dystrophic mice and can be included in a group of genes (again for RT-PCR or immunodetection). Refer to the figure. The level of CDKN1c / p57kip2 is significantly and reproducibly reduced in both dystrophin-deficient and caveolin-deficient mouse embryos compared to wild type. (B) pAkt rises with mdx and is suppressed in cav-3 ^{− / −} mouse embryos. FIG. 6 shows depletion of Pax-7 myoblasts in human skeletal DMD. FIG. 6 shows a graph showing depletion of Pax-7 myoblasts in human DMD and BMD compared to controls. Reduction of caveolin-3 levels to the wild type in mdx increases severity and sustains progression of the dystrophic phenotype in mdx mouse muscle. Effect of Igf-2 on dystrophic myoblasts; Pax-7 and Pax-3 are upregulated by Igf-2 treatment. Increased caveolin-3 levels in dystrophic myoblasts are enhanced by Igf-2 treatment.

Methods Mouse model Using wild-type (C57BL10) and isogenic mdx and cav-3 ^{− / −} mouse strains, cav-3 ^{− / −} dystrophic mice against C57B110 were found in Yoshito Hagiwara (Tokyo) (Hagiwara et al , 2000). Mdx and C57BL10 were generated in-house (Merrick et al., 2007). Double mutant mice (mdxcav +/−) are dystrophin null (dys − / −), caveolin-3 heterozygous (cav-3 +/−), and Igf-2 transgene heterozygous Crossing mdx and cav-3 ^{− / −} using previously described strategies to generate sex dystrophin-deficient mutants (mdxIgf−2 +/−; (Smith et al., 2000)) Generated by.
Genotyping is expressed by neomycin (to detect the cav-3KO transgene) and caveolin-3 (WT, mdx, and cav-3 ^+/− but not cav-3 ^{− / −} ; FIG. This was achieved by PCR in 7). The data shown is from two different littermates of each of E15.5 and E17.5 embryos containing approximately 50:50 ratios of mdx and mdxcav ^+/− embryos. Litter size (7-9 embryos per litter) was comparable to that obtained from WT and mdx.

Preparation of mouse embryos Wild-type (WT), cav-3 ^{− / −} , mdx, and mdxcav ^+/− embryos on the platform were fixed and treated as before and embedded in paraffin wax ( Smith and Merrick, 2008). The morning of plug detection was estimated to be E0.5. All sections were sagittal (5 μM). The plane and depth of the cut was established at the midline of the embryo (bisection point) using Kaufman's Atlas of embryology (Kaufman, 1992). Consistent wild type (WT), cav-3 ^{− / −} , mdx, and md from at least 3 separate embryos per line
xcav-3 ^+/− sections were used for all analyses.

Immunohistochemical methods Immunostaining and conditions for Igf-2, pan-myosin (MF20), fast muscle myosin heavy chain FMyHC (My32), and pax-7 antibody have been previously described (Merrick et al., 2007). CDNK1c / p57kip2 immunostaining has also been described previously (Westbury et al, 2001). pAkt detection was achieved at an optimal titer of 1/100 using an anti-pAkt rabbit polyclonal (No. 9277) obtained from New England Biolabs (UK) Ltd. . A long term peroxidase blocking step was included in all staining runs. The secondary antibody control showed unstained. B-cardiac myosin was detected using N2.261 antibody (1/1000; DHSB, Iowa City). CDNK1c / p57kip2, pAkt, IGF-2, and FMyHC staining, as described previously for FMyHC and IGF-2 (Merrick et al., 2007), percentage of antibody-positive myotubes in certain grid areas Was quantified by counting. Data were analyzed using Student's t test and ANOVA. At least 3000 myotubes were counted at each data point.

  Pax-7 immunostaining was also performed on stored young human muscle biopsies (see FIG. 8).

Analysis of myotube morphology and quantification in MF20 stained sections Sagittal MF20 stained embryos derived from wild type (WT), cav-3 ^{− / −} , and mdx
Matches were made in terms of section stage, plane, and angle. In order to avoid artifacts, we counted only the splits and branches that were completely at the cutting plane, which was carefully controlled between sections. This analysis may underestimate split / branch events. Sagittal sections from E13.5, E15.5, and E17.5 embryos are morphological to the published mouse atlas (Kaufman, 1995) at the midpoint of the embryo to facilitate coincidence The position of the scientific features was carefully matched. Sections were counted for both misalignment and branching phenotypes in a blinded manner and by two different observers and subjected to statistical analysis. For each mutant and wild-type embryo scored, a grid graduation was used to score branches in certain areas of the muscles that extend the same length in the intercostal, upper and lower extremity muscles, and facial muscle areas. . Scoring of misaligned fibers was achieved by orienting the muscle fiber direction of the longitudinal section with respect to the grid scale and scoring fibers that deviated beyond an angle of 25 degrees in a certain area. We have two experimenters counting slides, the second experimenter is unaware of the lineage, the split / branch is specified only in mdx, and the data is statistically significant. Is a reliable indicator of split / branch. When counting myotubes, we did not attempt to determine all myotube ends (which is an impossible task with embryo sections), but instead three different embryos per line. Matched sections of the proximal, distal, intercostal, and deep back muscles were carefully counted over a certain area (total myotubes counted per line: 2-3000). This allowed us to evaluate the ratio of myonuclei to myotubes and the average number of myotubes per section. Data analysis: ANOVA and Student t test.

Embryo explants E11.5-E17.5 embryos were dissected to isolate areas rich in skeletal muscle cells. In all embryos, the head, spinal cord, and internal organs were removed. In later embryos (E15.5 to E17.5), the skin and cartilage / bone were removed. The tissue rich in muscle was microdissected into microexplants and cultured in microwells (Smith and Merrick, 2008; Smith and Schofield, 1994). WT, mdx, or cav ^{− / −} E11.5 explant conditioned medium (CM) is removed from confluent culture, filtered (0.2 μm Acrodisc® syringe filter; VWR International, UK), standard Medium supplement (20% FCS; 2 mM glutamine) was supplemented and added to fresh E11.5 wild type explants and cultured for 18 days (Smith and Schofield, 1997). 180 explants from 3 embryos were analyzed for each of the 3 lines included in this study. Data was analyzed 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 confluence level of the cells in each well. Using Myf-5 immunostaining (Santa Cruz; titer 1/5000, rabbit anti-Myf-5 c20), E11.5-E17.5
Demonstrate muscle origin of wild type (WT), mdx, and cav-3 ^{− / −} explant cultures, 85%
More than 5 cells were myf-5 + (Smith and Merrick, 2008). Secondary antibody controls performed with each section always stained negative. Myf-5 c20 is used to extensively demonstrate myogenesis (Frock et al., 2006; Lindon et al., 1998).

Measurement of Apoptosis and Proliferation Confluent explant cultures with morphological characteristics of myoblasts were subcultured with dispase (Smith and Schofield, 1994) and seeded on coverslips (5 ×) before fixation. 10 ³ cells / cm ² ). Apoptotic nuclei cells were stained with 10 μg / mL DAPI for 3 minutes (Smith et al., 1995). Proliferating cells were immunostained as described above with Ki67 (rabbit anti-Ki67, titer 1/1000: manufactured by Novocastra Laboratories Ltd., UK). For antigen recovery (using a pressure cooker), a cover glass was first firmly mounted on the glass slide using a standard paper clip. Three separate experiments were performed in duplicate for each line (C57BL10, cav-3 ^{− / −} , mdx).

Protein isolation and immunoblotting Proteins were extracted directly from embryos and placed in glass homogenizers (1-5 mL; VWR International, UK) containing RIPA buffer. Immunoblots were performed using standard protocols and ECL (Pierce Endogene).
n Hyclone). Antibody: fast muscle myosin (My32, 1: 1000); α-tubulin (1: 1000, manufactured by Sigma); pax-7 (1/1000); caveolin-3 (1/1000). Goat anti-mouse IgG-HRP (1: 2000, manufactured by Santa Cruz Biotechnology). Protein concentration was determined using the Bradford assay in an ELISA format (Merrick et al., 2007).

Pax-7 RT (reverse transcription) real-time PCR
Reverse transcription (RT) PCR was performed to quantify the amount of pax-7 mRNA in myoblasts isolated from wild type (WT) and mdx mouse skeletal muscle (Smith & Schofield, 1994). To obtain quantitative data, linearization and equalization of the Pax-7 product was performed as described for Igf-2 (Merrick et al, 2007). RT-PCR under these quantifications,
It was performed on 6 separate isolates of wild type and 6 isolates of mdx myoblasts.
Polymerase chain reaction (PCR) cDNA samples were generated by the RT-PCR method described in Merrick et al, (2007) and subjected to PCR in the following reagent mixture:
・ 5 μL of 5 × PCR buffer (Promega)
・ 1.5 μL of 25 mM MgCl ₂ (Promega)
-5 μL of 10 mM dNTP (manufactured by Promega).
• 1 μL of 25 μM primer mix, 12 in PCR dH ₂ O (Sigma)
. 5 μM forward and 12.5 μM reverse primers (manufactured by Alta Biosciences).
1 μL of cDNA
1 μL Taq (Promega)-last added reagent

  Taq polymerase was added last to the PCR mixture (as above) to prevent random transcription. The sample was loaded onto a programmable thermal controller, which was specifically programmed for this gene as follows:

Primer sequence:
pax7-forward: gct-acc-agt-aca-gcc-agt-atg
pax7-reverse direction: gtc-act-aag-cat-ggg-tag-atg

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

Results Branching, fiber misalignment, and malformed myotubes characterize the embryonic phenotype of muscular dystrophy Wild-type (WT), cav-3 ^{− / −} , and mdx mice were immunostained with pan-myosin antibody (MF20) to produce muscle The fiber structure was revealed (FIG. 1). Bifurcation and fiber division are found on the mdx axis (back), subaxis (limb muscles, respiratory muscles), and facial muscles of E13.5-E17.5, which are early events related to myotube formation A dynamic temporal and spatial pattern consistent with certain is shown (FIGS. 1-1A-C and G). In the matched stages and muscle groups, mdx myotubes are dysplastic in dystrophic embryos compared to wild type, cav 3 ^{− / −} myotubes are hypertrophic, and myotube widths vary (FIG. 1− 1C-E, blue bar). The largest morphological defect occurs earlier in the mdx intercostal space (E13.5) than in the proximal limb (E15.5) and is consistent with the late development of these muscles (FIG. 1-1A). In cav-3 ^{− / −} and wild-type embryonic myotubes, there is no fiber splitting (FIGS. 1-1A,
C, E, and F, H). Myotube alignment is also impaired early in mdx myogenesis (FIGS. 1-2I). At E13.5, up to 1/8 of the mdx myotubes are misplaced from the midline fiber alignment of the proximal limbs, intercostals, and facial muscles (FIGS. 1-2I). This is statistically different from E13.5 wild type and cav-3 ^{− / −} muscles, where less than 5% of myotubes were misplaced. Myotubes at 25 ° from midline
Classifying more than half of misaligned E13.5 mdx myotubes (7-9%) when misclassified into less than 25 ° (misalignment) and less than 25 ° misplaced (tangential fibers) Aligned (FIGS. 1-2K) and the rest were tangential, but all misplaced myotubes were tangentially misplaced in wild-type or cav-3 ^{− / −} muscles. This was also true for the other periods studied (data not shown). The proportion of misplaced myotubes decreases with gestational age and depends on the muscle group (FIGS. 1-2I). At E13.5, 12% of the myotubes of the mdx intercostal muscles are misaligned, which decreases with gestational age, but we have found that muscles in the mdx intercostal muscles at all gestational stages Pipe misalignment and tangential misplacement were found (E13.5-E17.5, FIGS. 1-2I, M). WT and cav-3 ^{− / −} intercostal myotubes in the same phase are precisely aligned to the midline, and WT or c
There was no myotube deviation between the av-3 ^{− / −} intercostals, suggesting that the alignment mechanism of these muscles is tightly controlled and severely impaired in mdx (FIGS. 1-2H-N).

Morphological defects in dystrophic embryonic heart Cardiomyopathy is an important clinical consequence of both LGMD (1C) and DMD. We used MF20 and the myocardial β-myosin specific antibody N2.261 to demonstrate the morphology and myosin localization pattern of dystrophic and wild-type embryonic hearts (FIG. 2). Consistent with the literature, fast skeletal muscle myosin isoform (FMyHC) could not be detected in E13.5-E17.5 embryonic heart (My32 antibody, data not shown). Myocardial β-myosin is present in E13.5-E17.5 wild type and dystrophic ventricle and atrial myocytes (FIGS. 2-1A-F, FIGS. 2-2I-P). Both mutants show ventricular wall thickening (FIGS. 2-1A-F) and myocardial cell tissue disruption, which is much worse in cav-3 ^{− / −} and formed in a manner that myocytes crossed. However, they overlap each other (FIGS. 2-1E to F and GH). Atrial trabecularization is also impaired in dystrophic embryos, as opposed to long and hooked mdx trabeculae, short and thick in E14.5 cav-3 ^{− / −} embryos (FIGS. 2-2I˜ J)
. At E17.5, in contrast to the wild-type atrium where the staining is uniform and the two layers are tightly adjacent (FIGS. 2-2K, N), There is separation and loss of myocardial β-myosin (FIGS. 2-2K-P). There is a clear difference between the two dystrophic hearts, E17.5 cav-3 ^{− / −} has uneven and reduced myocardial wall staining, most notable in the trabeculae (FIG. 2-2O). ). In mdx, the loss of staining spreads over the intertrabecular region of the myocardium, but is largely retained at the end of the trabeculae (FIG. 2-2P).

Myonuclear misplacement and fusion abnormalities In E15.5 wild-type embryos, myonuclei are evenly spaced along the length of the myotubes, except for the new myotubes, evenly and spirally around the edges Are arranged in a shape (FIG. 3A, white star). In mdx, myonuclei are more centrally located than the wild type and are slightly further apart (FIG. 3-1B). In contrast, cav-3 ^{− / −} myonuclei are closer to each other, exhibiting severely impaired myonuclear spacing and nucleus “clustering”, which is particularly prominent at the end of the myotube (FIGS. 3-1C to D). The cross section demonstrates the presence of peripheral and central nuclei at this stage (E15.5) in wild-type muscle, and only the central nucleus is present in mdx and cav-3 ^{− / −} muscles ( FIGS. 3-1E-G). In order to quantify these phenotypes, we scored the sum of myonuclei and myofibers in a given area, and cav-3 ^{− / −} myotubes are either mdx or wild type myotubes. It was demonstrated that it contained twice myonuclei, which is statistically significant at E13.5, E15.5, and E17.5 (FIGS. 3-2K; p <0.05). In these phases, cav-3 ^{− / −} also has a significant excess of myotubes (p <0.05) and the number of myotubes of mdx is slightly but significantly less (p <0.05) (FIGS. 3-1H to J, FIGS. 3-2L). The imbalance in myotube numbers in cav-3 ^{− / −} embryos and mdx embryos decreases from E13.5 to E17.5, but is statistically different from wild type at all stages.

Hyperproliferation and apoptosis of cultured embryonic muscle stem cells To characterize embryonic muscle stem cell populations, we used dystrophic and wild type (E11.5-E17.5) embryonic myoblast cultures (Smith and Merrick, 2008). ). The somite origin of these cells was demonstrated using myf-5 staining (FIG. 4). Both mdx and cav-3 ^{− / −} explants show different outgrowth rates from wild type (WT) (FIGS. 4-1A).
~ B). Compared to the wild type, the outgrowth of myf-5 + cells was significantly greater in all phases (E11.5 to E17.5) in mdx and E15.5 to E17.5 in cav-3 ^{− / −.} large. At an earlier stage (E11.5 to E13.5), the cav-3 ^{− / −} outgrowth rate is indistinguishable from the wild type, suggesting that the defect occurs later in this mutant. Both myoblast proliferation (ki67 immunoreactivity) and apoptosis (Dapi staining) were significantly increased with mdx from E11.5 to E17.5, whereas in cav-3 ^{− / −} the apoptosis and proliferation rate was Both parameters are the same as wild-type apoptosis and proliferation rates up to E15.5, where they jump to levels approaching that of mdx embryos (FIGS. 4-1C, 4-2E). In mdx, proliferation (and apoptosis) remains significantly higher than wild-type levels, but decreases from E13.3 to E17.5, however, cav-3 ^{− / −} myoblast proliferation and apoptosis are , Increase to E15.5 (
FIGS. 4-1C to D and FIGS. 4-2E to F). At E17.5, the growth rate of cav-3 ^{− / −} is md
Although slightly reduced in line with x levels, the apoptosis rate is maintained at the level of E15.5 cav-3 ^{− / −} myoblasts (FIG. 4-2E). Soluble factor growth factor from mdx explants increases the outgrowth of E11.5WT explants (FIGS. 4-1I).

The pax-7 skeletal muscle stem cell population is reduced and tissue disrupted in dystrophic embryos At E17.5, the pax-7 ⁺ stem cell population is less densely distributed among skeletal muscle myotubes, but pax-7 Protein continues to increase with muscle size (Merrick et al., 2007); FIGS. 5-1A-B, GH, M and O). In wild-type embryos, the intensity of pax-7 staining is uniform and constant regardless of gestational age (FIGS. 5-1A-B, GH, and Q; low proximal limb). In E15.5 to E17.5, cav-3 ^{− / −} and mdx both cause a decrease in their pax-7 ⁺ cell population throughout their muscle tissue, and thus in both mutants E17. Pax-7 staining and pax-7 protein are significantly reduced up to 5 (FIGS. 5-1C-L, M, P; shown for low proximal limbs). This is particularly evident in cav-3 ^{− / −} proximal hind limb muscles, where pax-7 positive cells are scarcely present at E17.5 and their remaining staining is very weak (FIG. 5-1J, white Arrow). In E15.5 (cav-3 ^{− / −} and mdx) and E17.5 (mdx), pax-7 positive cell fragments were found in dystrophic muscle (FIG. 5-1L, indicated by black arrows), these This suggests that the cell may have undergone apoptosis. Dystrophin deficient (mdx), caveolin-3 was heterozygous (cav-3 ^+/-) mutant embryos (mdxcav- ^+/-) indicates a severe phenotype very proximal limb, Pax-7 positive cells are present at very low density in E15.5 (FIGS. 5-1MN and R), and almost no E17.5 is present in the low proximal limb (FIG. 5-
1O-P), it is greatly reduced across muscle tissue (see FIG. 7).

Whole embryo immunoblot demonstrated increased caveolin-3 protein in E15.5-E17.5 WT (and mdx) embryos compared to E11.5 and 13.5, cav-3 ^{− / -} with confirms that caveolin-3 protein is not present (Fig 5-1M~N).
In mdx, caveolin-3 was not detected in E11.5 (FIG. 5-1MN), as measured by densitometry on four separate gel runs, but caveolin- from the WT of E13.5 to E17.5. 3 content increases several fold (p = 0.05 for all three periods; FIG. 5-1O). Pax-7 content increases with pregnancy in wild-type embryos, but significantly decreases in cav-3 ^{− / −} and mdx of E15.5 to E17.5, and the decrease is cav-3 over mdx in both stages. Greater than ^-/- suggests that caveolin-3 may regulate pax-7 ⁺ myoblast survival in late pregnancy. At E15.5, the pax-7 band intensity of mdx and cav-3 ^{− / −} dystrophic embryos is reduced by 15% and 60%, respectively (FIGS. 5-1N, P). In E13.5, pax-7 is cav-3 ^- / - At slightly elevated, reduced in mdx, which, in this phase cav-3 ^- / - are found respectively in the embryo and mdx embryos It may be related to an increase or decrease in the number of myotubes (see Figure 3), and the timing of development suggests that these embryos may be damaged.

Mislocalization of fast muscle myosin isoforms in dystrophic embryos follows a reciprocal pattern in caveolin-3 and dystrophin-deficient embryonic muscles In the wild type, FMyHC is present in a small number of secondary myotubes as early as E11.5. By E15.5, it is strongly localized in about 45% of the secondary myotubes over a wide range of muscle groups. About 80% of myotubes contain FMyHC at E17.5, but the staining intensity is heterogeneous and weak in many myotubes (Merrick et al., 2007). This dynamic, muscle-specific fast muscle myosin localization pattern is impaired in cav-3 ^{− / −} and mdx mutant embryos, the timing of the occurrence of FMyHC staining, the existing fast muscle myosin positive myotube And abnormalities related to the intensity of fast muscle myosin staining (FIG. 6, FIG. S1). At E13.5, FMyHC rises above WT in some cav-3 ^{− / −} muscles (prominent in respiratory muscles), but all mdx muscles and ca
v-3 ^{− / −} is significantly decreased in the proximal muscle (FIGS. 6-1A to F).

Appearance of FMyHC ⁺ myotubes in mdx embryos mdx embryos and E13.5 delayed, the same period WT or cav-3 ^- / - as compared to the embryos, FMyHC ⁺ myotubes very throughout the musculature There are a few (respiratory muscles, FIGS. 6-1A-F; proximal muscles show a similar phenotype (not shown)). This finding was confirmed by immunoblotting in which the intensity of the mdx E13.5 band was statistically significantly reduced (FIGS. 6-2T and U), and the FMyHC ⁺ myotubes of E13.5 mdx embryos were statistically significantly (p <0.05) Supported by myotube counting (Figure 6-2V) demonstrating deficiency. A total of 6 mdx and 4 WT E13.5 embryos were used to demonstrate these data. In mdx, the staining intensity and rate of FMyHC-positive myotubes is greatly increased, so from E15.5 to E17.5, My32 staining of dystrophin-deficient myotubes is significantly excessive compared to the wild type, The staining intensity of the FMyHC band of the embryo immunoblot is increased (FIGS. 6-1G to O and FIGS. 6-2T). Increased mdx embryo FMyHC content and cav-3 ^{− / −}
This conclusion was confirmed by densitometry that revealed a decrease in embryonic FMyHC content. This is statistically significant, with P <0.001 and p <0.05, respectively (FIG. 6-2U). Quantification of matched FMyHC immunostained muscle sections of wild type and dystrophic embryos demonstrated that the percentage of FMyHC positive myotubes was perturbed in cav-3 ^{− / −} and mdx embryos (FIGS. 6-2V ). In mdx embryos (mdxcav-3 ^+/- ) in which cav-3 is heterozygous, E15.5 (FIG. 6-1R) and E17.5 (FIGS. 6-1Q, S)
Even in muscles such as the intercostal muscles (Figure 7-2T-U) where myotubes are significantly depleted, FMyHC was not down-regulated and increased FMyHC staining throughout the muscle tissue.

Extensive intercostal muscle fiber loss and pax-7 depletion in mdxcav ^+/- mutant embryos Upregulated caveolin-3 is likely to be in remission or contribute to the mdx / DMD phenotype In order to demonstrate whether the dystrophin was deficient (mdx) and the caveolin-3 null mutation was heterozygous (cav-3 ^+/- ) double abrupt Mutant embryos were generated. At E15.5, these embryos show a 50% reduction in caveolin-3 compared to wild type embryos (FIG. 5). By immunostaining with MF20 and pax-7, in E17.5, mdxcav-3 ^+/− embryos showed pax-7 positive intercostal muscles in addition to the loss of hindlimb pax-7 myoblasts (see FIG. 5). It is demonstrated to show a more severe phenotype, including a significant depletion of their intercostal muscle fibers with a significant reduction in blasts (FIGS. 7-1A-J). The fiber loss phenotype can be distinguished even at low magnification by the presence of interstices (“white space”) between mdxcav ^+/− intercostal fibers that are closely packed together in wild type intercostal muscle fibers ( FIGS. 7-1A, D, and F, I). At higher magnification (FIGS. 7-2K-P), this phenotype indicates that there are far fewer fiber clusters compared to wild type (WT), mdx, or cav-3 ^+/− intercostal muscles. The result is clear. This phenotype is very consistent, showing three different mdxcav ^+/− embryos (FIGS. 7-2N-P; md
xcav-3 ^+/- 1, 2, and 3). Even in mdx, intercostal fibers are thought to be distributed at a lower density than the wild type, suggesting a milder form of this phenotype. Quantification of fiber density was achieved by counting the number of intercostal fibers in a constant grid area, demonstrating that both mdx and het (mdxcav-3 ^+/− ) showed a decrease in fiber density.
This is a statistically significant reduction for the het mutant (p <0.05; FIG. 7-2R). Expressed as a percentage of wild type, mdxcav-3 ^+/− mutant embryos show 71.2% massive depletion of their intercostal myotubes by E17.5 (FIGS. 7-2S; 3 different E17.5 mdxcav-3 ^+/- average number of embryos), it can be seen that mdx shows a third (37.5%) depletion. The majority of these fibers express fast muscle myosin (FMyHC) in both mutants (Figure 7-2U).

Pax-7 staining of a human muscle biopsy This is shown as FIG. 8 and demonstrates that young human muscle can be stained in a manner similar to that described above.

Pax-7 gene expression studies Pax-7 mRNA levels were quantified using RT-PCR. This showed that the DM-7 individuals had lower pax-7 levels than normal individuals.

Igf-2 impaired mouse
Merrick et al (2007) describes Igf-2 in muscle fibers. FIG. 9 shows Igf-2 in a disabled mouse. Igf-2 message (mRNA) is elevated in mdx and cow-3 ^{− / −} embryos. Inhibition of Igf-2 in caveolin-deficient embryos is at the post-transcriptional level.

A decrease in the IgF-2 downstream gene CDKN1 / P57kp2 or altered expression of pAkt (phosphorylated Akt) is characteristic of muscular dystrophy, as shown by the expression levels in FIG. pAkt shows an MD disease type specific response, for example, pAkt is reduced in caveolin-3 deficiency / LGMD-1c compared to normal muscle and increased in mdx / DMD. The failure pattern of pAkt is the same as that of Igf-2.

DISCUSSION In this study, we demonstrated the embryonic phenotype of mdx and cav-3 ^{− / −} dystrophic mice, and the mouse phenotype of both proteins (mdxcav-3 ^+/− ) Indicates significantly more severe. We have demonstrated the important role of dystrophin and caveolin-3 in mouse embryonic development, and the pathology defining the adult dystrophic phenotype and clinical picture of DMD and LGMD-1c stems from embryonic muscle differentiation failure Prove that. The key findings of this analysis are summarized in Table 2.

Early muscular patterning is impaired in dystrophin-deficient embryos Myf-5 ⁺ mdx embryo myoblast proliferation behavior is impaired from E11.5, mdx
myf-5 + myoblasts are hyperproliferative and apoptotic. Myf-5 characterizes the embryonic myoblast population and is expressed in the muscle plate before myotube differentiation is initiated (E8.5) and is found in all muscle groups throughout myogenesis (Hadchouel et al. al., 2003; Ott et al., 1991). Dystrophin is one day after Myf-5 is expressed at E8.5, with fully differentiated muscularis (on-axis) myotubes first appearing and myosin heavy chain (MyHC) first expressed 24 It is expressed at an important stage (E9.5) during muscle plate differentiation, before time (E10.5) (Houzelstein et al., 1992; Ott et al., 1991; Schofield et al., 1993). Early ventral and secondary myogenesis is due to the later appearance of FMyHC, pax-7, and cav-3 proteins, and incomplete formation and tissue collapse of mdx muscle tissue in E11.5-E13.5 Is severely damaged and delayed in mdx embryos as shown by (Fig. 1; Fig. 6 and ancillary data Fig. S1). These data indicate that dystrophin has an essential role in muscle plate differentiation, and when muscle plate differentiation is impaired in mdx embryos, it has serious consequences for patterning and function of the entire muscle tissue I suggest that.

Also at E10.5, myf-5 + myoblasts migrate from the muscle plate and initiate ventral myogenesis (Cusella-De Angelis et al., 1992). Myoblasts migrate under the control of the hox genes (pax3 and lbx) and differentiate in response to growth factors (wnt1 and shh) secreted by adjacent tissues (Gross et al., 2000; Hadchouel et al. , 2003). The cues that initiate embryonic myoblast migration are unknown, and in other tissues, stem cell migration is controlled by inductive cues from the source and target tissues and is essential for accurate patterning of embryonic structures, It can be easily impaired (Lehmann, 2001). Consider E10.5-11.5 axial myotubes to trigger myf-5 + myoblast migration from the muscle plate and provide signaling cues that initiate ventral and secondary myogenesis. Is possible. In the absence of dystrophin, these cues are not present and both migration and initiation processes are hindered. The abnormal behavior of E11.5 wild-type explants cultured in E11.5 mdxCM (FIGS. 4-1I) shows that the E11.5 muscle plate is a secreted factor that modifies the behavior of the E11.5 embryo myoblast population. Support the view of releasing. Impairment of early myogenesis in mdx suggests that these initial role (s) of dystrophin are different from those of utrophin. This conclusion suggests that in vitro dystrophin and utrophin published mRNAs that are exclusive in the early embryonic stage
Consistent with the situ pattern (Houzelstein et al., 1992; Schofield et al., 1993). At later stages of pregnancy (see below), mdx myogenesis is “catch-up” that can be mediated by subsequent overproduction of caveolin-3 by these embryos, or by compensatory expression of another protein, such as a-utrophin. There seems to be a process. In postnatal muscle, a-utrophin is up-regulated in dystrophin-deficient muscles and mdx can take on several functions of dystrophin (Weir et al., 2004). Due to the increased severity of the double mutant phenotype (mdxcav-3 ^+/- ) in late pregnancy, the compensatory effect of caveolin-3 in mdx embryos in late pregnancy is also claimed.

Caveolin-3 is regulated by muscle regulator (MRF), activated during myotube differentiation, and expressed later in dystrophin in the E11.5 muscle plate (Biederer et al., 2000).
. This is consistent with our detection of caveolin-3 in E11.5 wild-type embryos. MRF also controls pax-7, which also appears at E11.5 (Merrick et al., 2007). In cav-3 ^{− / −} embryos, the initial muscle patterning process appears to be intact, myf-5 + myoblast behavior is not impaired, and localization and timing of ventral myogenesis is wild. Equivalent to type. Therefore, caveolin-3 is not considered necessary for these initial myogenesis. In mdx, the appearance of caveolin-3 and pax-7 is both delayed, which may be a consequence of delayed development of myogenesis in these embryos, or more specific downstream of dystrophin inactivation.

Ventral and secondary myogenesis Interpreting the role of dystrophin in late embryonic events is complicated by the upregulation of caveolin-3 in E13.5 to mdx embryos. In postnatal tissues, overexpression of caveolin-3 causes dystrophin downregulation and a DMD-like phenotype (Galbiati et al., 2000). Loss of dystrophin causes DGC disassembly and suppression of dystrophin-related proteins (Ohlendieck et al., 1993; Vaghy et al., 1998). However, DMD patients and adult mdx muscles have 1.5-2.4 and 2-3 fold excess caveolin-3, respectively (Vaghy et al., 1998). In E13.5-E17.5 mdx embryos, we found an average (phase-dependent) 3-5 fold excess of caveolin-3, and impaired caveolin-3 expression was early in dystrophin deficiency. This is an important consequence. The dystrophin embryonic mRNA expression pattern overlaps that of wild-type dystroglycan, but not utrophin, and this pattern is unchanged in mdx embryos (Houzelstein et al., 1992; Schofield et al., 1995) ). Thus, dystrophin can negatively regulate caveolin-3 by binding competitively to β-dystroglycan (Ilsley et al., 2002).

In zebrafish embryos, caveolin-3 mutants show cytoskeletal and fusion abnormalities, and dystrophin mutants show unstable muscle binding, suggesting a role in embryonic myotube placement and orientation ( Bassett et al., 2003; Nixon et al., 2005). E13.5 myogenesis is impaired at cav-3 ^{− / −} and mdx, and the phenotype suggests that mouse caveolin-3 and dystrophin have a similar role as the corresponding substances in zebrafish. In E13.5 mdx, we found myotube misplacement, splitting, and bifurcation. Abnormal myotube orientation is not found in caveolin-3 deficient mutants. Instead, embryonic cav-3 ^{− / −} myonuclei are clustered at the end of the myotube, suggesting a T-tube abnormality reported postnatally in cav-3 ^{− / −} (Minetti et al., 2002). Excess cav-3 ^{− / −} myotube production from E13.5 to E17.5 was also found to double the myocardial content and hypertrophy. These data are consistent with in vitro study data using cultured cav-3 ^{− / −} myoblasts,
In this study, a reciprocal phenotype (reduced myotube number, fewer myonuclei, and hypertrophy) was found in caveolin-3 overexpressing myoblasts, and caveolin-3 suppresses myoblast fusion. This suggests (Volonte et al., 2003). E13.5 mdx embryos partially reproduce an overexpression phenotype that shows a reduction in myotubes and the number of myotubes. The number of myonuclei is not affected. However, at the late stage (E15.5-E17.5), caveolin-3 levels remain high, but the number of myotubes of mdx is comparable to the wild type. This is because caveolin-3 can control myotube size, but E13.5 myotube abnormalities are not due to excessive caveolin-3 but due to a delay in mdx ventral myogenesis. (See item above).

Reciprocal impairment of fast muscle fiber specification in cav-3 ^{− / −} and mdx FMyHC + myotubes first appear at E11.5 in wild-type abdominal muscle and predict the onset of secondary myogenesis (Merrick et al., 2007). Fiber type switching begins at E15.5, where several myotubes switch between slow and fast muscle myosin expression, and the process of demonstrating the adult fiber type ratio begins (Cho et al., 1994; Merrick et al. , 2007). In mdx, FMyHC + myotube differentiation is markedly impaired (see above), and fast muscle fiber type specification is impaired in both mdx and cav-3 ^{− / −} (FIG. 6). In E15.5 to E17.5, m
There is a reciprocal phenotype where dx has a significantly excess FMyHC protein and a higher proportion of FMyHC + myotubes compared to WT, and in cav-3 ^{− / −} FMyHC is greatly reduced. Overproduction of FMyHC may be due to a “catch-up process” in E13.5 mdx muscle, which is facilitated by a compensatory increase in caveolin-3. However, loss of FMYHC + myotubes in cav-3 ^{− / −} at E15.5 to 17.5 requires caveolin-3 also for fast muscle fiber specification, and overproduction of caveolin-3 in mdx The type is pathogenic and suggests that it causes overproduction of FMyHC fibers in the mdx of E15.5 to E17.5. Postnatal fast muscle fibers preferentially degenerate in DMD and mdx, resulting in severe consequences for muscle function (Webster et al., 1988). In a mutant that is heterozygous for caveolin-3 but completely deficient in dystrophin (mdxcav-3 ^+/− ), the FMyHC + fiber is E17. Compared to the wild type (WT).
5 still exists in excess, suggesting that this is a balanced relationship between dystrophin and caveolin-3 that is important for the exact rate of fast muscle fibers, rather than caveolin-3 levels alone.

In adult tissues, caveolin-3 and dystrophin interact directly in DGC by competitive binding of β-dystroglycan (Sotgia et al., 2000). These data demonstrate that dystrophin and caveolin play distinct roles in myogenesis, but suggest that both proteins and functional DGC may be required for fiber type specification.

Loss of pax-7 myoblasts cav-3 ^{− / −} and mdx of E15.5 to E17.5 indicate depletion of pax-7 ⁺ myoblasts and significant depletion of pax-7 protein. The loss of pax-7 occurs more rapidly at cav-3 ^{− / −} of E15.5, which is the time point when caveolin-3 is strongly upregulated in wild-type embryos, and this loss is more than that of E15.5 mdx. large. Caveolin-3 can induce survival signaling in muscle, similar to pax-7 itself. Our data suggest that caveolin-3 plays a direct role in the rapid loss of pax-7 + myoblasts at cav-3 ^{− / −} of E15.5, pax-7 + muscle Blast depletion suggests that their increased caveolin-3 levels may be partially compensated in mdx muscle (Relaix et al., 2006; Smythe et al., 2003). This conclusion indicates that in the mdxcav ^+/− mutant, pax-7 is significantly more depleted than any single mutant, so that the pax-7 cells are mdxcav ^+/− low until E17.5. It is not present at all in the proximal limb muscles, but is supported by being greatly reduced in other muscles. Pax-7 ⁺ myoblasts are important for normal postnatal satellite cell appearance (Relaix et al., 2006; Seale et al., 2000).
In the absence of pax-7 (pax-7 ^{− / −} mice), satellite cells have a reduced cell number,
Apoptosis increases. However, because of the presence of pax-3 + myoblasts, satellite cells are not completely lost, although there may be enough satellite cells to achieve and sustain the (postnatal) juvenile muscle development of these mice. Adult regeneration is impeded (Oustanina et al., 2004; Relaix et al., 2006; Seale et al., 2000). E17.5 mdxca
The finding that v ^+/- embryonic intercostal muscle is significantly depleted of myotubes as well as pax-7 + cells
It suggests that ax-7 may play an important role in embryonic muscle formation. These suggest that dystrophic embryos reduce their ability to generate muscle fibers from late pregnancy that can be passed on to life after birth. Dystrophy muscle regeneration is known to be abnormal and is associated with myoblast apoptosis, hyperproliferation, irregular fiber size, and progressive fiber deposition. Both mdx and cav-3 ^{− / −} muscles experience their muscle degeneration and the onset of regeneration associated with high levels of apoptosis in the early postnatal week. (Hagiwara et al., 2000; Smith et al., 1995) In mdx, failure of muscle regeneration proceeds from the fourth month, which may be partially mediated by upregulation of utrophin in mdx. Mitigated by simultaneous reduction of the denaturation process, but not by DMD (
Reimann et al., 2000; Roig et al., 2004; Roma et al., 2004). Regeneration has not been studied in detail in adult cav-3 ^{− / −} . According to the data, the regeneration mechanism of dystrophic mutants is abnormal at birth because these mutants are deficient in pax-7, and this early loss is later advanced in the regenerative process of the adulthood This suggests that it may underlie sexual disorders. The significant loss of E17.5 mdxcav ^+/- embryonic respiratory muscle muscle fiber density, along with massive loss of pax-7 + cells, further reinforces this conclusion, and increased caveolin-3 levels indicate that mdx Suggests an important compensatory role in the degenerative phenotype (and potentially in the early stages of DMD).

Correlation between dystrophic mouse phenotype and clinical pathology of MD DMD and LGMD 1c are early-onset and progressive pediatric skeletal muscle diseases that affect myocardial and skeletal muscle function and muscle stability (Hoffman et al. , 1987; Minetti et al., 2002). DMD is a wide range of progressive dysfunction of whole muscle tissue, abnormal caveolin-3 expression, myoblast apoptosis, cardiomyopathy, and regenerative abnormalities (Cox and Kunkel, 1997; Smith et
al., 1995; Vaghy et al., 1998).

LGMD-1C exhibits similar but limited myopathy changes, particularly affecting the limbs, diaphragm and heart muscle (Galbiati et al., 2001; Hagiwara et al., 2000; Smythe et al. al., 2003). The postnatal phenotypes of mdx and cav-3 ^{− / −} are sufficiently similar in most respects to the clinical pathology of DMD and LGMD and are 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). Respective embryo phenotypes of mdx and cav-3 ^{− / −} , as models of LGMD and DMD, reinforce the validity of these mice, to the underlying mechanisms of MD and the mode of function of dystrophin and caveolin-3 It provides new insights and suggests new approaches to review the differences between the human and murine forms of the disease. We identify important developmental stages; dystrophin and caveolin-3 each play an important role, revealing two new pathologies in late pregnancy, muscle plate differentiation (E11.5) and secondary Myogenesis (E13.5); Fast muscle fiber specification and pax-7 myoblasts that provide insights into the mechanisms underlying MD pathology and suggest a novel pathway for therapeutic intervention and early diagnosis of MD Exhaustion.

Cardiomyopathy, particularly left ventricular failure, is an important clinical consequence of DMD and many other MDs, and is a proven pathology of cav-3 ^{− / −} , mdx, and caveolin-3 overexpressing 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 are expressed in E9.5 and E10.5 mouse embryonic hearts, respectively (Biederer et al., 2000; Houzelstein et al., 1992). At E13.5, mdx has moderate thickening of the apex and atrial trabecular abnormalities. cav-3 ^{− / −} has markedly ventricular thickening, atrial abnormalities, and progressively worsening myocyte organization characteristic of cardiomyopathy, early onset in 3-4 months caveolin-3 deficient mice Consistent with sexual postnatal cardiomyopathy. These data indicate that caveolin-3 is required for normal heart development, has a role (s) in cardiomyopathy, and β-myocardial myosin is misguided in E17.5 mdx and cav-3 ^{− / −} It has been localized, trabecular abnormalities, β-myocardial myosin abnormalities underlie some well-known cardiomyopathy, thus suggesting that impaired expression in dystrophic hearts is important ( Cuda et al., 1993). Atrial disorders have not been previously reported in any mouse. This finding on mdx suggests that the recent report that trabeculae are over-formed in 28-year-old DMD is derived from development (Finsterer et al., 2005). Postnatal mdx hearts have been reported to show WT levels of β-myocardial myosin and increased utrophin levels that can compensate for dystrophin deficiency, but these mice have progressive cardiomyopathy (Quinlan et al., 2004). The localization of β-myocardial myosin has not yet been demonstrated after birth, so its atrial localization impairment may persist in adults or may be lost at birth or at an early age. Yes (Wilding et al., 2005).

Hyperproliferation and increased myocyte apoptosis are well documented postnatal MD muscle pathology characterizing both the mouse and human forms of DMD / mdx and LGMD-1c / cav-3 ^{− / −} and 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 a role in survival signaling (Glass, 2005; Smythe et al., 2003). In mdx muscle, high levels of myoblast apoptosis and myoblast hyperproliferation are observed from 1 week after birth to adulthood (Smith, 1996; Smith et al., 1995; Spencer
et al., 1997) and (in this study) demonstrated in the embryonic stage of E11.5-E17.5.
These data suggest impaired stem cell behavior, and apoptosis is an important contributor to early consequences of dystrophin loss and DMD pathology. Myoblast apoptosis later occurs at cav-3 ^{− / −} of E15.5, suggesting that this phenotype may be an accurate predictor of disease severity.

New Insights-Importance to MD These data present a new perspective on the pathogenesis of muscular dystrophy, demonstrate the role of embryos for dystrophin and caveolin-3, which advance understanding of myogenesis, pax-7 myoblasts It suggests that therapeutic strategies that attempt to replace cells and modify fiber type specification may be productive in the initial treatment of muscular dystrophy. Early diagnosis and therapeutic intervention are likely to improve the quality of life of patients with muscular dystrophy. It is further suggested that caveolin-3, fast muscle myosin, Igf-2, CDKN1c / P57kip2, and pAkt are used in the assay.

Data from further diagnostic assays We performed pax-7 staining on human skeletal muscle samples as follows: control (non-disease) children, 31 days of age (FIGS. 12A-C); 16 weeks (FIG. 12D). -F) and 12.7 weeks (FIGS. 12G-I) (non-disease) human fetal tissue. These data demonstrate that the Pax-7 staining pattern in normal fetal and neonatal skeletal muscle is directly comparable (equivalent) to that found in mouse embryos and neonatal tissues at the same stage. Pax-7 + myoblasts are abundant in both human and mouse fetal and neonatal tissues, are uniformly distributed, and are strongly labeled with the pax-7 antibody. We also investigated the localization of pax-7 positive myoblasts in 10 different (non-muscular dystrophy) muscle diseases, and in each of these diseases pax-7 positive myoblasts are abundant, The number of non-diseased muscle samples was not significantly different. A representative sample (myotubule myopathy) is shown in FIGS. 12 (J-L). However, the results were different for skeletal muscle samples from Duchenne and Becker muscular dystrophy (DMD and BMD) patients (FIGS. 12M-P and FIG. 13). In DMD and BMD, the number of pax-7 positive myoblasts was significantly depleted compared to the control, consistent with disease severity (DMD muscle was less mild in Pax-7 cells. It was more consistently depleted than the Becker type of form). Pax-7 staining intensity was also reduced compared to normal samples, which was particularly evident in DMD patient muscles with a low number of Pax-7 positive myoblasts, predominantly with a slight expression of Pax-7. DMD patient muscles did not contain any pax-7 positive myoblasts in large areas (FIGS. 12M-P, weakly stained pax-7 + cells and areas without Pax-7 + cells are indicated by asterisks ^*. ing). These data demonstrate that pax-7 depletion is an early marker of DMD and BMD that can be used to distinguish these diseases from other non-MD myopathy and pediatric non-disease muscle biopsies. This marker can be used alone or as a marker of muscle degradation (eg, creatine kinase) and / or a marker of muscle regeneration (eg, developing myosin, Igf-2) and / or other markers of the disease (eg, , Caveolin-3, fast muscle myosin) before the onset of overt clinical signs (DM
D can identify muscular dystrophy of fetuses, newborns, and young children before birth and from birth to 5 years of age and younger). These data also strongly support the use of Pax-7 alone or in combination with other markers as markers of therapeutic efficacy and as indicators of disease progression.

Further treatment data Caveolin-3 is elevated above normal (wild-type) levels in mdx (mouse) embryos and postnatal tissues, and DMD (human) muscle. Double mutant mouse embryos that are deficient in dystrophin (mdx) and heterozygous for caveolin-3 have reduced levels of caveolin-3 and more severe muscles than those found only in dystrophin deficiency (mdx) It shows pathology and suggests that high levels of caveolin-3 can compensate for the loss of dystrophin. FIG. 14 shows that when caveolin-3 levels were reduced and returned to non-disease (wild-type) levels in mdx mice, the mdx postnatal dystrophy phenotype also increased in severity and increased caveolin-3 levels in these mice The therapeutic effect is confirmed (FIG. 14). Using Western blotting, we consistently compared the level of caveolin-3 protein found in these (DM-double mutant) animals compared to that found only in mdx mice. It was reduced and demonstrated to be comparable to the level found in wild type mice (FIG. 14A). At the histological level, dystrophic pathology in mdx mice is most severe at 3-8 weeks after birth. Importantly, the progression of the disease persists in dystrophin-deficient mice with wild-type levels of caveolin-3 compared to mdx and compared to mdx muscle of the same age (9 weeks of age) or Compared to the milder pathology found in caveolin-3 ^{− / −} mice (cav-3 ^{− / −} ) (FIG. 14D-E), DM (het) muscle 8 weeks later (FIGS. 14B-C) Progressive severe pathology persists in Taken together, these data identify that increased caveolin-3 protein levels have a therapeutic effect on dystrophin-deficient pathology and caveolin-3 upregulation is an effective treatment for Duchenne muscular dystrophy.

Igf-2 exhibits a remission effect on the dystrophic (mdx) phenotype (Smith et al, 2000), but the mechanism is unknown. FIG. 15 demonstrates that Igf-2 can upregulate the expression of Pax-7, Pax-3, and caveolin-3 in dystrophic myoblasts, suggesting a direct mechanism of its therapeutic effect Provide strong support for using this protein as a therapeutic agent separately or in conjunction with other therapies such as caveolin-3 (FIG. 15). (AB) Igf-2 induces increased expression of Pax-7 (mRNA) in wild-type (WT) and dystrophic myoblasts within 15 minutes treated with 10-20 μg / mL Igf-2 . This effect persists and Pax-7 continues to be upregulated in myoblasts until at least 24 hours (statistically significant, p <0.01, student t test). (CD) Similarly, Igf-2 induces up-regulation of Pax-3 message within 15 minutes, which lasts until at least 24 hours (20 μg / mL Igf-2: p <0 .01). (EF) Diffusion response curves of Igf-2 treatment of Wt myoblasts, Igf-2 also induces the growth of pax-7 protein (here, p <0, indicating 8 hours after treatment) .01). (G) The ratio of caveolin-3 protein expression in wild type (WT) and dystrophic myoblasts, as measured by Western blotting, exceeds the level where caveolin-3 in dystrophic myoblasts is found in the wild type. 2 to 3 times as an example. Within 15 minutes, Igf-2 treatment induces up to 8-fold increase in caveolin-3 protein in dystrophin myoblasts compared to wild type, which lasts until at least 24 hours (p <0). .01, 20 μg / mL Igf-2).

Claims (20)

  A method for identifying an individual exhibiting symptoms of muscular dystrophy, for monitoring the treatment or progression of muscular dystrophy, or identifying an individual having a propensity to develop muscular dystrophy, in a tissue sample from said individual , Determining the expression level of one or more proteins selected from pax-7, caveolin-3, and / or fast muscle myosin.   The method of claim 1, wherein pax-7 is measured.   The method according to claim 1 or 2, wherein caveolin-3 is measured.   A method according to any preceding claim, wherein fast muscle myosin is measured.   The method according to any of the preceding claims, further comprising determining one or more expression levels of insulin-like growth factor-2 (Igf-2), CDKN1c / P57kip2, and / or pAkt in the tissue sample. Method.   The method according to claim 1, wherein the muscular dystrophy is Duchenne muscular dystrophy (DMD) and / or limb girdle muscular dystrophy (LGMD).   The method according to any of the preceding claims, wherein the tissue sample is derived from the infant before or after birth.   The method according to any of the preceding claims, wherein the expression level of the protein is determined by immunoassay. One or more expression levels of the protein are
(I) by immunostaining the sample and / or (ii) by immunoblotting of the protein,
9. The method of claim 8, comprising determining.   The method according to any one of claims 1 to 7, wherein the expression level of each protein is determined by quantitative polymerase chain reaction (QPCR).   Encodes two or more antibodies specific for two or more of pax-7, caveolin-3, and / or fast muscle myosin, or two or more of pax-7, caveolin-3, and / or fast muscle myosin A kit for use in a method according to any of the preceding claims comprising two or more pairs of PCR primers specific for mRNA.   The kit according to claim 11, wherein the protein is pax-7 protein or mRNA encoding pax-7.   The kit according to claim 11 or 12, wherein the protein is caveolin-3 or mRNA encoding caveolin-3.   The kit according to claim 11, wherein the protein is fast muscle myosin or mRNA encoding fast muscle myosin. A kit for use in the method according to claims 1 to 7, comprising an antibody specific for pax-7 protein together with paraformaldehyde.   The kit according to claims 11 to 15, comprising an antibody specific for Igf-2 or a PCR primer pair.   17. Kit according to claims 11-16, comprising one or more reagents for tyramide signal amplification (TSA).   A method of treating muscular dystrophy comprising administering caveolin-3 to a patient or increasing the patient's caveolin-3 expression.   A pharmaceutical formulation comprising caveolin-3 in combination with a pharmaceutically acceptable carrier.   Caveolin-3 used to treat muscular dystrophy.

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