KR20160092201A - A composition for treating muscle damage disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating muscle damage diseases, which comprises an AKAP6 (A Kinase Anchoring Protein 6) expression increasing agent as an active ingredient. According to the present invention, by identifying new factors involved in muscle regeneration and muscle regeneration, a method of effectively treating muscle damage disease targeted therewith can be provided.

Description

[0001] The present invention relates to a composition for treating muscle injury diseases,

The present invention relates to a pharmaceutical composition for preventing or treating muscle damage diseases, which comprises an AKAP6 (A Kinase Anchoring Protein 6) expression increasing agent as an active ingredient.

Skeletal muscle regeneration is an ongoing phenomenon to restore muscles damaged by exercise, chronic diseases, trauma, and the like. Such regeneration of skeletal muscle is regulated by Myogenic Regulatory Factor (MRF) such as MyoD, Myf5, Myogenin and MRF4, but the regulatory factors of these MRFs have not yet been clarified.

In addition, the development process of the skeletal muscle is also regulated by the MRF family. MyoD and Myf5 need to be upregulated for specific differentiation from mesodermal progenitor cells to myogenic lineage during embryogenesis . Proliferating myoblast cells eventually differentiate into myogenin and MRF4 expressing myocytes and then express muscle specific genes such as myosin heavy chain (MyHC). Differentiated mononuclear cells fuse with each other to form polynuclear canal / myofibers. Mice lacking both MyoD and Myf5 are known to die as soon as they are born due to the absence of skeletal muscle. Mieogenin mutant mice have been reported to be difficult to survive before and after childbirth due to a decrease in skeletal muscle. However, since the MyoD or Myf5 single-mutant mouse is known to have no muscle damage, myogenin is considered to be an important factor in muscle development.

On the other hand, a scaffold protein is a protein that functions as a platform to efficiently interact with signaling enzymes involved in signaling and partner molecules. A well-known family of such scaffold proteins is AKAP (AK Kinase Anchoring Protein), which acts as a scaffold for protein kinase A and their substrates.

In particular, AKAP6 / mAKAP is highly expressed in the heart, skeletal muscle, and brain and has been reported to decrease expression in MDX mouse, a muscle dystrophy mouse. It is known to regulate cardiac function, but skeletal myocytes And the function of AKAP6 in skeletal muscle regeneration is unknown.

The present inventors have made intensive studies to develop a method for treating muscular injuries that target the new factors involved in muscle regeneration and muscle regeneration. As a result, AKAP6 and myogenin have a mutual positive feedback action, Myogenin promotes muscle regeneration by increasing the expression of AKAP6, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating muscle damage diseases, which comprises myogenin as an active ingredient as an agent for increasing expression of AKAP6 (A Kinase Anchoring Protein 6).

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating muscle damage diseases, which comprises an AKAP6 (A Kinase Anchoring Protein 6) expression increasing agent as an active ingredient.

In addition, the present invention provides a method of preventing or treating a muscular damage disease, comprising the step of administering to a subject an AKAP6 expression increasing agent.

The present invention also provides a use of an AKAP6 expression increasing agent for the prophylaxis or treatment of muscular dysfunction.

In one embodiment of the present invention, the expression-increasing agent is a Myogenin gene or a protein.

In another embodiment of the present invention, the myogen gene is characterized in that it is composed of the nucleotide sequence of SEQ ID NO: 1.

In another embodiment of the present invention, the myogen protein is characterized in that it is composed of the amino acid sequence of SEQ ID NO: 2.

In another embodiment of the present invention, the muscular dystrophy is selected from the group consisting of muscular dystrophy, muscular atrophy, myositis, polymyositis, peripheral vascular disease, and fibrosis fibrosis). < / RTI >

According to the present invention, by identifying new factors involved in muscle regeneration and muscle regeneration, it can be used as a target to detect muscular dystrophy, muscular atrophy, myositis, polymyositis, A method and an application for efficiently treating a muscle injury disease such as peripheral vascular disease and fibrosis can be provided.

Figure 1 shows the results of immunofluorescence staining (A) and Western blotting (B) to determine whether AKAP6 promotes differentiation of mouse stem cells (C2C12).
Figure 2 shows the results of Western blotting and RT-PCR (mRNA) to select differentiation markers that are associated with increased expression of AKAP6 during myoblast differentiation.
Figure 3 shows the results of immunofluorescent staining to further confirm that myogenin (Figure 3a) and MyHC (myosin heavy chain) (Figure 3b) are differentiation markers of myocytes.
Figure 4 shows the results of immunofluorescence staining to confirm whether AKAP6 promotes differentiation of human skeletal muscle stem cells (HSMM).
FIG. 5 shows Western blotting (protein) and RT-PCR (mRNA) in order to determine the effect of AKAP6 knockdown on mouse myocytes (C2C12) on the expression of differentiation markers, myogenin and MyHC to be.
FIG. 6 shows the results of immunofluorescence staining to determine the effect of knockdown of AKAP6 on mouse myocytes (C2C12) and the expression of myogenin (FIG. 6A) and MyHC (FIG. 6B), which are differentiation markers.
FIG. 7 shows immuno-fluorescence staining (A) and Western blotting (B) to determine the effect of knockdown of AKAP6 on the expression of myogenin and MyHC in human skeletal muscle cell (HSMM) Results.
FIG. 8 is a graph showing changes in expression of AKAP6 by knocking down myogenin (siMyoG), Western blotting (protein) and RT-PCR (mRNA) in order to confirm the correlation between myogenin and AKAP6 The result is confirmed.
FIG. 9 is a graph showing changes in AKAP6 expression by Western blotting (protein) and RT-PCR (mRNA) after overexpressing myogenin (pMyoG) to confirm the correlation between myogenin and AKAP6 The result is confirmed.
Fig. 10 shows the result of analysis of the nucleotide sequence of the AKAP6 promoter.
FIG. 11 shows the result of confirming that the site where myogenin binds directly to E-box 3 among 7 E-boxes in the AKAP6 promoter through ChIP assay.
Fig. 12 is a result of confirming that E-box3 is a site where myogenin directly binds among 7 E-boxes in AKAP6 promoter through Luciferase assay.
Fig. 13 shows the result of confirming myofiber regeneration process by histo-staining (H & E) and immunofluorescence (fluorescence) in an animal model of cardiotoxin damage to CTX (cardiotoxin).
FIG. 14 shows the results of Western blotting of changes in AKAP6 expression during myofiber regeneration in an animal model of cardiotoxin (CTX) muscle injury.
Fig. 15 shows the function of the AKAP 6 in the invisible playback in in- vivo ). To further confirm this, we injected AKAP6 shRNA into muscle-damaging mouse animal models and knocked down AKAP6.
Fig. 16 shows the result of immunofluorescence staining to assess the expression of laminin 2 present in the normal skeletal muscle and evaluate whether muscle tissue regeneration with shAKAP6 is possible.
17 is a result of performing a RotaRoad experiment on CTX muscle injured mice in order to functionally identify the role of AKAP6 in muscle regeneration.
18 is a schematic diagram showing the positive feedback of AKAP6 and myogenin during the muscle regeneration process.

The present inventors observed AKAP6 expression during mouse / human myocardial differentiation under the assumption that the AKAP family is an important regulator of skeletal muscle myoblast differentiation and muscle regeneration. As a result, the expression of AKAP6 gradually increases with differentiation Respectively.

In addition, in the present invention, when AKAP6 is knocked down (inhibited) using siRNA, the formation of myotube is inhibited due to no differentiation despite the differentiation condition, and miogenin, which is an important transcription factor of muscle differentiation, is also inhibited Respectively.

Further, in the present invention, in order to clarify the correlation between AKAP6 and myogenin, inhibition of myogenin inhibits AKAP6 expression and overexpression of myogenin increases AKAP6 expression, This is the first study to show positive feedback between the two proteins. Specifically, in order to verify this, the AKAP6 promoter was cloned and it was confirmed that the site binding to myogenin was E-box3, and that the transcription factor, myogenin, increased the expression of AKAP6.

In addition, in the present invention, in order to further confirm the function of AKAP6 in vivo in the muscle regeneration process, shAKAP6 lentivirus was prepared, and the muscle damage model of the mouse was established and the function of AKAP6 was verified. Specifically, in order to produce a cardiotoxin (CTX) injury model, which is a representative animal model of muscle repair caused by muscle damage, shControl and shAKAP6 were injected into the mouse tibialis anterior muscle and CTX was injected one week later to injure the muscles. After a certain period of time, a rotarod analysis was performed to confirm the degree of muscle regeneration. This is to measure the running time by placing the mouse on the spinning cylinder. In the shakap6 injected group, the muscle regeneration was suppressed and the running time was remarkably reduced.

These results indicate that AKAP6 increases myogenin and myogenin also increases AKAP6 and promotes muscle regeneration through mutually positive feedback. As a result, it is known that AKAP6 increases myogenin Is expected to be useful for the treatment of myopathy.

The key role of the scaffold protein is to physically assemble the signaling related factors so that they interact well with partner molecules. A typical scaffold protein, AKAP, is an upstream activator and a downstream effector Are assembled in the same macromolecular complex. Because myopathies and muscle regeneration are processes that require a high degree of regulation by a variety of regulators, myogenin upregulates the AKAP6 scaffold to efficiently control the signaling cascade of muscle differentiation and muscle regeneration I think.

As used herein, the expression enhancer is not particularly limited as long as it significantly increases the expression of the target gene, but it is preferably a miogenin gene or protein as an expression enhancer of AKAP6 (A Kinase Anchoring Protein 6).

As used herein, the term " muscle damage disorder "refers to any disease in which muscle tissue or muscle cells are damaged by damage to muscular tissue, such as muscular dystrophy, muscular atrophy, Myositis, polymyositis, peripheral vascular disease, and fibrosis.

In the present specification, "composition for preventing or treating muscular injury disease" is used in the same sense as "composition for muscle regeneration ".

The compositions of the present invention may further comprise components such as conventional therapeutically active ingredients, other adjuvants, pharmaceutically acceptable carriers, and the like. The pharmaceutically acceptable carrier includes saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and the like.

The term "individual" as used herein refers to a subject in need of treatment for a disease, and more specifically refers to a human or non-human primate, mouse, rat, dog, cat, It means mammals.

The term "pharmaceutically effective amount" as used herein refers to the amount and severity of the disease to be treated, the age and sex of the patient, sensitivity to the drug, administration time, administration route and rate of release, Can be readily determined by those skilled in the art in a quantity that is determined by well-known factors in the art and can be maximized without adverse effects taking all of the factors into consideration.

The composition of the present invention is not limited as long as it can reach the target tissues. For example, oral administration, arterial injection, intravenous injection, percutaneous injection, intranasal administration, transbronchial administration, or intramuscular administration. The daily dose is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, and is preferably administered once a day or divided into several times a day.

The composition of the present invention can be used variously for medicines, foods and beverages for prevention and improvement of muscle damage diseases, and can be used in the form of powders, granules, tablets, capsules or drinks.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  1: Experimental method

1-1. Induction of myoblast cell culture and differentiation

Mouse C2C12 myoblast (ATCC: CRL-1772) was cultured in Dulbecco's modified Eagle's medium (GIBCO) containing 10% FBS (Lonza) and 1% penicillin / streptomycin (GIBCO) cells were cultured in differentiation medium (DMEM supplemented with 2% horse serum and 1% penicillin / streptomycin) to induce differentiation into myotubes.

Human HSMM human myoblast cells (Lonza) were cultured in SkGM-2 medium (Lonza) and then replaced with DMEM / F12 (Lonza) containing 2% horse serum for induction of differentiation.

Human kidney cells HEK 293A and HEK 293T cells (ATCC) were maintained in DMEM (GIBCO) containing 10% FBS (Lonza) and 1% penicillin / streptomycin (GIBCO).

1-2. Immunofluorescence  dyeing

Mouse C2C12 source cells or human HSMM source cells were seeded in μ-Dish ^{35 mm high} (ibidi), cultured until 70-80% confluence, and then replaced with differentiation medium. After differentiation, the cells were fixed in 4% PFA for 10 min at 4 ° C and blocked with blocking buffer (2% BSA-PBS) for 1 hour and then incubated with anti-AKAP6 (Covance), anti-myogenin (Santa Cruz) Or anti-MyHC (Sigma) overnight at 4 < 0 > C. Subsequently, fluorescent dyes were sequentially labeled with a secondary antibody (Invitrogen) to which they were attached. Nuclei were stained with DAPI (Molecular Probe) and mounted using a fluorescent mounting medium (DAKO). Fluorescence images were obtained using a confocal microscope (Carl Zeiss LSM710).

1-3. siRNA  And an expression vector transfection

For AKAP6 or myogenin-specific knock-down, siRNA binding to each mRNA was introduced into cells with Metafetamin pro (Biotex) 24 hours before induction of cell differentiation.

Here, both the siRNA for AKAP6 and the scrambled siRNA (non-targeting siRNA pool) as a control group were purchased from Dharmacon, and the sequence of AKAP6 specific siRNA was as follows.

AKAP6 siRNA: 5'-GACGAACCUUCCUUCCGAAUU-3 '

In addition, siRNA for myogenin and scrambled siRNA (non-targeting siRNA pool) as a control were purchased from Santa Cruz.

The myogenin full-length cDNA was PCR-amplified using the following primer pairs, cloned into pcDNA 3.1 (Invitrogen) vector, and introduced into cells with Metafetamin pro (Biotex).

Forward primer: 5 '- (HindIII) GGGAAGCTTATGGAGCTGTATGAGA-3'

Reverse primer: 5 '- (EcoRI) CGGGAATTCTCAGTTGGGCAT-3'

1-4. Western Blotting  analysis

Cells were washed with PBS buffer and lysed with RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP 40, 0.1% SDS, 0.5% deoxycholate, Protease Inhibitor Cocktail (Roche)). 20 μg of total protein was electrophoresed on SDS-PAGE, transferred to PVDF membrane, reacted with primary antibody, and incubated with horseradish peroxidase-linked secondary antibody for 1 hour. At this time, using α- tubulin (Calbiochem) as an internal control, and the band was measured with a ^{Novex ® ECL Chemiluminescent Substrate Reagent (Invitrogen} ).

1-5. RNA  Separation and RT - PCR

RNA was extracted from cells with Trizol reagent (Invitrogen) and RT-PCR was performed at 42 ° C for 1 hour using Prime first reverse transcriptase kit (Takara) to obtain cDNA. The obtained cDNA was electrophoresed on 1.5% agarose gel to measure the mRNA expression level, and the control group was standardized and quantified using GADPH. The primer pairs used for RT-PCR are shown in Table 1 below.

RT-PCR primer Sequence (5 '-> 3') AKAP6 Forward TCTGGGGACATAAGTGTGAG Reverse CCTGAATGATGCGTTGGACT Myogenin Forward GCGCAGGCTCAAGAAAGTGAAT Reverse GTTGAAGTCGCAGGAGACAAC MyoD Forward CATCCGCTACATCGAAGGTC Reverse TCGCATTGGGGTTTGAGCC MyHC Forward AGAAGGAGGAGGCAACTTCTG Reverse ACATACTCATTGCCGACCTTG GAPDH Forward CATGACAACTTTGGCATTGTG Reverse GTTGAAGTCGCAGGAGACAAC

1-6. Chromatin Immune sedimentation ( Chromatin immunoprecipitation ; ChIP ) analysis

Chromatin immunoprecipitation (ChIP) analysis for myogenin was performed using the ChIP assay kit (Millipore) according to the manufacturer's instructions.

After transformation with the mouse AKAP6 promoter vector, cell lysates were immunoprecipitated with anti-myogenin antibody (Santa Cruz).

PCR was performed with the primer pairs shown in Table 2 below in order to amplify the AKAP6 promoter region having seven myogenin protein binding regions.

ChIP primer Sequence (5 '-> 3') E-box 1 Forward GCTACTAACCCTGAATACACAG Reverse CTATCCAGCCTTCCACAGAG E-box 2 Forward CACACTGGAAAGAAAGGACTG Reverse GTTCTGGGGTTAAAATCTGG E-box 3 Forward CCAGATTTTAACCCCAGAAG Reverse AGTGCACAGACTAATAATCG E-box 4-5 Forward TCCTGAAGGTTAAGTGGTAG Reverse CCCTATCAGGCAATTTGATC E-box 6 Forward ACTAGCCAGGGAGAAGAGCGATCA Reverse GCTGCATTTCCAGTGGAGCCT E-box 7 Forward GTAGAGCAGCAAACGAAGAGG Reverse CCTTGTTTGACCTGCTGCATG

1-7. Promoter building and Luciferase assay

The AKAP6 promoter structure was obtained from genomic DNA of C57 mouse. The 1.7 kb upstream sequence of the transcription initiation site of AKAP6 gene was amplified by PCR and cloned into pGL3-basic luciferase vector (Promega).

HEK 293A cells were then plated per well in each of 3 x 10 ⁵ cells in 6-well plate at a density, transformed with the desired plasmid. After transformation, cells were lysed with Reporter Lysis Buffer (Promega), and luciferase assay was performed using Luciferase Assay System kit (Promega) and Luminometer (Promega). Transformation efficiency normalization was determined by β-galactosidase enzyme assay system (Promega).

1-8. CTX ( cardiotoxin ) - Muscle injury animal model and muscle tissue staining

All animal studies were conducted in accordance with the approval of the National Institute of Animal Science and Ethics (IACUC) and the NIH Guidelines for the Use and Management of Laboratory Animals.

First, male C57BL / 6 (8-10 weeks old) was anesthetized and 50 μl of CTX (10 μM, Sigma) or saline (control) was administered to the TA tibialis muscle of each leg of each mouse Respectively. Total tibialis was collected at 1, 3, 5, 10, 14 days after CTX injection and embedded in OCT compound or paraffin. The embryonic tissues were cut into 4-6 μm and stained with Hematoxylin & Eosin (Sigma) or incubated with primary anti-AKAP6 and anti -laminin α2 (Alexis Biochemical) antibodies, followed by secondary antibody conjugated with fluorescent dye (Invitrogen) Lt; / RTI >

1-9. Lentivirus  Produce

Animal model ( in In order to knock down AKAP6 in vivo , AKAP6 shRNA was packaged as lentivirus and injected into an animal. The sequence of AKAP6 shRNA is as follows.

AKAP6 shRNA: 5'-GACGAACCTTCCTTCCGAATTCAAGAGATTCGGAAGGAAGGTTCGTCTTTTT-3 '

Specifically, AKAP6 shRNA was cloned into a pLL3.7 vector (Addgene), and pLp1, pLp2 and pLp3 plasmids (Invitrogen) were prepared by using polyethyleneimine (PolyEthylenImine; PEI, Polyscience) Were transfected into HEK293T cells with pLL3.7-GFP or pLL3.7-AKAP6. After 2 days, the supernatant transfected was collected and ultracentrifuged at 25,000 rpm for 90 minutes at 4 < 0 > C. The concentrated lentiviral pellet was resuspended in PBS and 50 [mu] l of lentivirus was injected into the mouse tibialis anterior (TA).

1-10. Rotarod  Experiment

In order to observe the athletic ability of the mice after induction of muscle injury, a Panlab Rota-Rods LE. 8200, Harvard Apparatus experiment was performed.

First, mice were injected with control shRNA or AKAP6 shRNA lentivirus 7 days before CTX injection. Two weeks later, the mouse was placed on a rotating rod and the latency time from the rotating rod to the falling mouse under continuous acceleration (5-40 rpm) was measured to examine the motor function. Each mouse was subjected to three experiments to measure the respective delay times and calculate the average delay time. Between each experiment the mice were allowed to rest for more than 5 minutes.

1-11. Statistical analysis

Quantification of band intensities was performed with Image J software (NIH, Bethesda, MD, USA) and standardized for internal control intensity. Experimental results are expressed as mean ± standard deviation (SD). Differences in each group were compared by unpaired t-test or Mann-Whitney U-test. P <0.05 was considered statistically significant.

Example  2: AKAP6 Myocytes myoblast ) Identification Promotion of Differentiation

The following experiment was conducted to investigate the group involved in myoblast differentiation among the AKAP family (AKAP6, AKAP12, AKAP-Lbc, AKAP79) which is known to be associated with skeletal muscle.

First, as a result of culturing the mouse root cells (C2C12) and inducing differentiation into myotubes, the cell morphology was observed. As shown in Fig. 1 (A), the myotube And more than 80% of the cells were differentiated into canaliculus cells on the fourth day.

In addition, Western blotting was carried out by extracting proteins from the respective cells. As a result, as shown in Fig. 1B, only the AKAP6 expression increased during the differentiation and the remaining AKAP12, AKAP-Lbc and AKAP79 increased cell proliferation And no difference was observed between the differentiation.

Example  3: source cells Differentiation marker  Confirm

Since skeletal muscle stem cells are known to be involved in various transcription factors (TF) such as MyoD, myogenin, MyHC and the like, the expression pattern of AKAP6 has no correlation with the transcription factors .

3-1. Mouse root cells ( C2C12 ) Experiment

First, protein and RNA were extracted from each mouse C2C12 cell obtained in Example 2, and Western blotting and RT-PCR were carried out. As a result, as shown in FIG. 2, AKAP6 And expression of Myogenin and MyHC was increased with increasing expression.

These results imply that myogenin and MyHC are likely to be differentiation markers. To further confirm this, we performed immunofluorescent staining for AKAP6, myogenin and MyHC. As a result, as shown in FIGS. 3A and 3B, during the progress of differentiation, red fluorescence for AKAP6 was observed in the nuclear envelope, and myogenin and MyHC were also observed as green fluorescence in the nucleus in which AKAP6 was expressed .

3-2. Human myoblasts ( HSMM ) Experiment

Immunofluorescent staining was carried out in the same manner as in Example 3-1 except that human skeletal muscle root cells (HSMM) were used instead of mouse cells. As a result, as shown in FIG. 4, red fluorescence for AKAP6 was significantly increased during differentiation.

Example  4: AKAP6 Knockdown  Effect verification ( in vitro )

AKAP6 was knocked down under cell differentiation conditions using AKAP6 siRNA (5'-GACGAACCUUCCUUCCGAAUU-3 ') to further confirm the function of AKAP6 in source cell differentiation.

4-1. Mouse root cells ( C2C12 ) Experiment

As shown in Fig. 5, AKAP6 knockdown in the mouse root cell C2C12 resulted in reduction of AKAP6, which was up-expressed during differentiation, at both mRNA and protein levels in AKAP6 siRNA treatment as compared with control (siCon) treatment. At the same time, the proteins of myogenin and MyHC, which are myelogenous markers, were decreased, but mRNA reduction was confirmed only in myogenin.

As a result of immunofluorescence staining, it was confirmed that myogenin and MyHC (green fluorescence) also decreased together with decrease of AKAP6 (red fluorescence) which was increased during differentiation as shown in Figs. 6A and 6B.

4-2. Human myoblasts ( HSMM ) Experiment

As a result of knockdown in human myoblast HSMM, the same results as those of the mouse cells of Example 4-1 were observed, as shown in Fig.

Example  5: Miogenerine and AKAP6 Interaction analysis

From the results of Examples 3 and 4, the myogenenin was selected as a final muscle differentiation marker related to AKAP6, and the correlation between myogenin and AKAP6 was examined more specifically.

Western blotting and RT-PCR were carried out by knocking down myogenin using siRNA (siMyoG). As shown in FIG. 8, AKAP6 was found to be expressed at mRNA and protein level Both were significantly degraded.

On the contrary, when Western blotting and RT-PCR were carried out after overexpression of myogenin using an expression vector pMyoG in which the myogenin gene was cloned, as shown in Fig. 9, AKAP6 Was significantly increased at both mRNA and protein levels.

Example  6: Miogenerine AKAP6  Promoter binding confirmation ( in vitro )

Through Example 5, it was confirmed that myogenin functions as an expression enhancer of AKAP6. Therefore, it was further investigated whether myogenin binds to the AKAP6 promoter and acts as a transcription factor.

First, the region estimated to be the AKAP6 promoter was identified using the ensemble program (www.ensembl.org), and the ~ 1.7 kb upstream sequence at the start of AKAP6 transcription was cloned and sequenced. As a result, it has been known that the helix-loop-helix MRFs family such as miogenin binds to the E-box sequence (CANNTG) in the promoter to promote muscle-specific gene expression. Actually, the AKAP6 promoter has seven E-box sequences (See Fig. 10).

6-1. ChIP assay

As shown in FIG. 11, the third E-box (E-box 3) was a binding site as a result of confirming the site where myogenin directly binds among seven E-boxes through the ChIP assay. On the other hand, it was confirmed that E-box 1 and E-box 3 have the same sequence (CATGTG) but do not bind to E-box 1.

6-2. Luciferase assay

Luciferase assay further proved that E-box 3 is a myogenin binding site. As shown in Fig. 12, after continuous deletion mutants were generated in the AKAP6 promoter, luciferase analysis was performed by overexpressing myogenin, and it was found that luciferase activity was increased in E-box 1 or E-box 2 At the time of deficiency, there was no significant difference from the control (wild type, WT), but it was significantly decreased at the time of deficiency of E-box 3.

Example  7: AKAP6 of Muscle regeneration  Induction Verification ( in vivo )

In order to confirm the role of AKAP6 in muscle regeneration, we examined the expression of AKAP6 during muscle regeneration using muscle injury model.

7-1. CTX  Muscle injury animal model and tissue staining

As shown in Fig. 13A, the histological staining (H & E) results of an animal model of muscle injury by CTX (cardiotoxin) showed that extensive recovery of muscle fibers (myofiber) .

7-2. Immunofluorescent staining ( immunofluorescence staining )

As a result of immunofluorescence with anti-AKAP6 antibody, AKAP6 (green fluorescence) was observed in the nuclear membrane of regenerated muscle fibers (->), whereas in the nuclear membrane of mature muscle fibers, Little was observed (▷)

These results are consistent with the result of FIG. 14, which confirmed the expression pattern of AKAP6 protein through Western blotting.

Example  8: AKAP6 Knockdown  Effect verification ( in vivo )

To further confirm the function of AKAP6 in muscle regeneration, AKAP6 shRNA was injected into mouse animal models ( in vivo ) to knock down AKAP6.

Specifically, GFP (Green Fluorescence Protein) -AKAP6 shRNA-containing lentivirus was prepared, injected into the TA muscle of the mouse, and treated with CTX to induce muscle injury. As a result, as shown in Fig. 15A, it was confirmed that lentivirus infection was successful because both shAKAP6-GFP and shMock-GFP (control group) were observed to have green fluorescence. In addition, as shown in Fig. 15B, in the shAKAP6-GFP-treated group, it was confirmed that the knockdown was also successful because the AKAP6 protein was significantly decreased as a result of Western blotting.

Since muscle fiber of skeletal muscle is surrounded by basal lamina and it is known that its main component is laminin, the expression of laminin a2, which exists in a large amount in normal skeletal muscle, was examined and evaluation of muscle tissue regeneration by shAKAP6 was evaluated. As a result, as shown in Fig. 16, the basal lamina surrounding all the skeletal muscle fibers was strongly and wholly homogeneously expressed by the anti-laminin alpha2 antibody in the muscle tissue infected with the control group shMock-GFP lentivirus (Arrow mark), and the red fluorescent signal was significantly reduced and heterogeneity in the muscle tissue infected with shAKAP6-GFP lentivirus (* mark).

Example  9: Rotarod  Evaluation of athletic performance in vivo )

In order to functionally identify the role of AKAP6 in muscle regeneration to restore muscle damage, a RotaRoad experiment was performed after CTX muscle damage.

As a result, as shown in Fig. 17, it was found that the mouse (CTX + shAK6) infected with shAKAP6-GFP lentivirus significantly decreased the exercise capacity as compared with the mouse (CTX + shM) infected with shMock-GFP lentivirus . These results are consistent with the data of Example 8, supporting the role of AKAP6 in muscle regeneration after muscle injury.

As shown in FIG. 18, AKAP6 increases myogenin and myogenin also increases AKAP6, and muscle regeneration is promoted by positive feedback between the two proteins, as shown in FIG. 18 have.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> SNU R & DB FOUNDATION <120> A composition for treating muscle damage disease <130> PB14-12402 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 675 <212> DNA <213> Human <220> <221> gene <222> (1). (675) <223> Myogenin <400> 1 atggagctgt atgagacatc cccctacttc taccaggaac cccgcttcta tgatggggaa 60 aactacctgc ctgtccacct ccagggcttc gaaccaccag gctacgagcg gacggagctc 120 accctgagcc ccgaggcccc agggcccctt gaggacaagg ggctggggac ccccgagcac 180 tgtccaggcc agtgcctgcc gtgggcgtgt aaggtgtgta agaggaagtc ggtgtccgtg 240 gaccggcggc gggcggccac actgagggag aagcgcaggc tcaagaaggt gaatgaggcc 300 ttcgaggccc tgaagagaag caccctgctc aaccccaacc agcggctgcc caaggtggag 360 atcctgcgca gtgccatcca gtacatcgag cgcctccagg ccctgctcag ctccctcaac 420 caggaggagc gtgacctccg ctaccggggc gggggcgggc cccagccagg ggtgcccagc 480 gaatgcagct ctcacagcgc ctcctgcagt ccagagtggg gcagtgcact ggagttcagc 540 gccaacccag gggatcatct gctcacggct gaccctacag atgcccacaa cctgcactcc 600 ctcacctcca tcgtggacag catcacagtg gaagatgtgt ctgtggcctt cccagatgaa 660 accatgccca actga 675 <210> 2 <211> 224 <212> PRT <213> Human <220> <221> PEPTIDE &Lt; 222 > (1) <223> Myogenin <400> 2 Met Glu Leu Tyr Glu Thr Ser Pro Tyr Phe Tyr Gln Glu Pro Arg Phe   1 5 10 15 Tyr Asp Gly Glu Asn Tyr Leu Pro Val His Leu Gln Gly Phe Glu Pro              20 25 30 Pro Gly Tyr Glu Arg Thr Glu Leu Thr Leu Ser Pro Glu Ala Pro Gly          35 40 45 Pro Leu Glu Asp Lys Gly Leu Gly Thr Pro Glu His Cys Pro Gly Gln      50 55 60 Cys Leu Pro Trp Ala Cys Lys Val Cys Lys Arg Lys Ser Val Ser Val  65 70 75 80 Asp Arg Arg Ala Ala Thr Leu Arg Glu Lys Arg Arg Leu Lys Lys                  85 90 95 Val Asn Glu Ala Phe Glu Ala Leu Lys Arg Ser Thr Leu Leu Asn Pro             100 105 110 Asn Gln Arg Leu Pro Lys Val Glu Ile Leu Arg Ser Ala Ile Gln Tyr         115 120 125 Ile Glu Arg Leu Gln Ala Leu Leu Ser Ser Leu Asn Gln Glu Glu Arg     130 135 140 Asp Leu Arg Tyr Arg Gly Gly Gly Gly Pro Gln Pro Gly Val Pro Ser 145 150 155 160 Glu Cys Ser Ser His Ser Ala Ser Cys Ser Pro Glu Trp Gly Ser Ala                 165 170 175 Leu Glu Phe Ser Ala Asn Pro Gly Asp His Leu Leu Thr Ala Asp Pro             180 185 190 Thr Asp His Asn Leu His Ser Leu Thr Ser Ile Val Asp Ser Ile         195 200 205 Thr Val Glu Asp Val Ser Val Ala Phe Pro Asp Glu Thr Met Pro Asn     210 215 220

Claims (5)

A pharmaceutical composition for preventing or treating muscular injury diseases, comprising an expression-increasing agent for AKAP6 (A Kinase Anchoring Protein 6) as an active ingredient. 2. The composition of claim 1, wherein the expression enhancer is a Myogenin gene or protein. 3. The composition of claim 2, wherein the myogen gene is comprised of the nucleotide sequence of SEQ ID NO: 1. 3. The composition of claim 2, wherein the myogen protein is comprised of the amino acid sequence of SEQ ID NO: 2. The method according to claim 1, wherein the muscular dystrophy is selected from the group consisting of muscular dystrophy, muscular atrophy, myositis, polymyositis, peripheral vascular disease, and fibrosis. &Lt; / RTI &gt;

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