Composition for preventing, alleviating, or treating muscle diseases, comprising NFAT inhibitor as active ingredient

Abstract

The present invention relates to a composition for preventing, alleviating, or treating muscle diseases, the composition comprising an NFAT inhibitor as an active ingredient. An NFAT inhibitor (VIVIT peptide) comprising the amino acid sequence of SEQ ID NO: 1 according to the present invention was found to: alleviate the inhibition of muscle differentiation by suppressing NFAT signaling pathway activation induced by excessive calcium exposure, and increase the expression levels of muscle differentiation-related proteins in C2C12 myoblasts and mouse muscle-derived myoblasts. Thus, the NFAT inhibitor can be effectively used as a composition for preventing, alleviating, or treating muscle diseases.

Description

Composition for preventing, improving or treating muscle disease containing NFAT inhibitor as an active ingredient

[0001] The present invention relates to a composition for preventing, improving or treating muscle disease, comprising an NFAT inhibitor (Nuclear Factor Activated T cell inhibitor) as an active ingredient.

[0002] Our body's muscles attach to bones, protect them, and perform various functions, including maintaining proper body shape. Muscles also promote calcium uptake, increasing bone density. However, as the body ages, changes in composition lead to a redistribution of body fat and protein. Around age 50, the rate of protein synthesis within muscle cells slows down compared to its breakdown, leading to rapid muscle degeneration and exposure to sarcopenic diseases.

[0003] Sarcopenia, a muscle-losing disorder, refers to a condition in which approximately 13-24% of the normal body mass is lost. Sarcopenia significantly reduces activity, which not only harms mental health but also reduces life satisfaction. It also makes it easier to injure oneself in everyday life, potentially leading to serious injuries. One of the causes of sarcopenia is the gradual decline in skeletal muscle mass and quality that occurs with aging, as well as weight loss, including fat and body fat components, due to inadequate dietary energy intake.

[0004] Myoblasts, formed through the division and determination of precursor cells, differentiate into muscle cells (myocytes) and then fuse with adjacent cells to form myotubes. Differentiated myotubes form myofibrils, forming muscles. Some myoblasts remain quiescent as stem satellite cells. When muscle tissue is exposed to trauma or diseases such as muscular dystrophy, muscle stem cells known as satellite cells repair and regenerate damaged muscles. In diseases like muscular dystrophy, these cells are particularly important for muscle stability and healing.

[0005] Meanwhile, NFAT (nuclear factor of activated T cells) is a cytoplasmic protein that is regulated through a calcium ion-dependent signaling pathway and translocates to the nucleus and becomes activated in response to changes in intracellular calcium concentration. NFAT protein is dephosphorylated by calcineurin, a calcium-activated phosphatase. The dephosphorylated NFAT then translocates to the nucleus and induces the transcription of several cytokine genes, including the IL-2 gene required for T cell activation. Generally, NFAT activation plays a crucial role in the induction of immune responses. However, excessive activation can lead to immunopathological responses, including autoimmunity, transplant rejection, and inflammation. Therefore, modulating NFAT transcription could be useful in the treatment of immune-related diseases.

[0006] As for NFAT-related technologies, Korean Patent No. 0863626 discloses a 'membrane-permeable NFAT inhibitory peptide', and Korean Patent No. 1603279 discloses a 'pharmaceutical composition for preventing or treating a disease related to the activity of NFAT5, containing a protoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient'. However, no composition for preventing, improving or treating a muscle disease, containing the NFAT inhibitor of the present invention as an active ingredient, has been disclosed yet.

[0007] The present invention was derived from the above-mentioned needs, and provides a composition for preventing, improving or treating muscle diseases, which contains an NFAT inhibitor as an active ingredient, and when the NFAT inhibitor (VIVIT peptide) consisting of the amino acid sequence of SEQ ID NO. 1 of the present invention is treated to myoblasts whose muscle differentiation is inhibited by calcium, the activation of the NFAT signaling system is inhibited, the inhibition of muscle differentiation is alleviated, and the expression level of muscle differentiation-related proteins is increased in C2C12 myoblast cell lines and mouse muscle-derived myoblasts, thereby completing the present invention.

[0008] To solve the above problem, the present invention provides a pharmaceutical composition for preventing or treating muscle disease, which contains an NFAT inhibitor (Nuclear Factor Activated T cell inhibitor) as an active ingredient.

[0009] In addition, the present invention provides a health functional food composition for promoting muscle growth, preventing or improving muscle loss or muscle strength loss, which comprises an NFAT inhibitor as an active ingredient.

[0010] In addition, the present invention provides a veterinary composition for preventing or treating muscle disease, comprising an NFAT inhibitor as an active ingredient.

[0011] In addition, the present invention provides a feed additive for promoting muscle growth, preventing or improving muscle loss or muscle strength loss, containing an NFAT inhibitor as an active ingredient.

[0012] The present invention relates to a composition for preventing, improving or treating muscle disease, comprising an NFAT inhibitor as an active ingredient, wherein the NFAT inhibitor (VIVIT peptide) consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention alleviates inhibition of muscle differentiation by inhibiting activation of the NFAT signaling system caused by excessive calcium exposure, and has an excellent effect of increasing the expression level of muscle differentiation-related proteins in C2C12 myoblast cell lines and mouse muscle-derived myoblasts.

[0013] Figure 1 shows the results of confirming the changes in myogenic differentiation in myoblasts continuously exposed to calcium. (A) is the result of immunofluorescence analysis of Myh3 expression in C2C12 myoblasts continuously exposed to calcium, (B) is the result of confirming the Myh3 and MyoG protein expression levels (left) and Myh3 mRNA expression levels (right) in C2C12 myoblasts continuously exposed to calcium, and (C) is the result of confirming the Myh3 and MyoG protein expression levels (left) and Myh3 mRNA expression levels (right) in mouse-derived primary myoblasts continuously exposed to calcium. ** indicates that Myh3 mRNA expression levels were significantly reduced in the calcium-treated group compared to the calcium-free group, p<0.01. GM represents cell growth medium, and DM represents differentiation medium. (D) is a schematic diagram of an experiment to induce muscle differentiation for muscle fiber formation in human embryonic stem cell-derived myoblasts, and iPCS represents induced pluripotent stem cells. (E) shows the results of confirming the protein expression level of MYH in human embryonic stem cell-derived myoblasts, and (F) shows the results of observing the size of human embryonic stem cell-derived myotubes by calcium exposure, and the graph on the right is a quantified graph. * indicates that the size of myotubes was significantly reduced in the calcium-treated group compared to the calcium-free group, p<0.05.

[0014] Figure 2 shows the results of RNA sequencing for transcriptome analysis of mouse muscle-derived myoblasts continuously exposed to calcium. (A) is a graph visualizing genes whose expression significantly changed in mouse muscle-derived myoblasts whose myogenesis was inhibited under calcium-treated conditions through transcriptome analysis using a volcano plot, (B) is a graph showing the number of genes (282) whose expression levels were significantly downregulated by more than twofold compared to the control group that was not continuously exposed to calcium, and (C) is a graph showing the number of genes (243) whose expression levels were significantly upregulated by more than twofold compared to the control group that was not continuously exposed to calcium (p<0.05).

[0015] Figure 3 shows the results of analyzing the growth of myoblasts under myogenic differentiation conditions by calcium exposure. (A) is the result of confirming the C2C12 myoblast cell line continuously exposed to calcium, and (B) is the result of confirming the result of confirming primary myoblasts derived from mouse muscle. Myoblast growth according to the concentration of calcium treatment was observed by staining the cell nucleus, quantified, and represented in a graph. *, ** indicate that cell growth significantly increased in the calcium-treated group compared to the calcium-free group; * indicates p<0.05, and ** indicates p<0.01. (C) is the result of confirming the protein expression level (left) and mRNA expression level (right) of Pax7, a growth indicator gene, in mouse muscle-derived myoblasts continuously exposed to calcium. * indicates that Pax7 mRNA expression level significantly increased in the calcium-treated group compared to the calcium-free group; p<0.05.

[0016] Figure 4(A) shows the results of GSEA (Gene set enrichment analysis) analysis of the NFATc1 signaling pathway, calcineurin signaling pathway, DNA replication (a marker of cell division), and muscle differentiation-related gene sets in mouse muscle-derived myoblasts continuously exposed to calcium. Figures 4(B) and 4(C) show the results of confirming the mRNA expression level of Ccnd1, an NFAT target gene, in C2C12 myoblast cell line (B) and mouse muscle-derived myoblasts (C) continuously exposed to calcium.

[0017] Figure 5 shows the results of confirming the changes in gene expression levels in the muscle tissues of patients with calcific tendonitis. (A) CCND1 (Cyclin D1), a growth indicator gene and NFAT signaling pathway target gene, (B) MYH3 (Myosin Heavy Chain 3), a muscle differentiation indicator gene, (C) SPRY1 (Sprouty RTK Signaling Antagonist 1), a myoblast resting phase indicator gene, and (D) PAX3 (paired box gene 3) are the results of confirming the mRNA expression levels. *, *** indicate that the mRNA expression levels of muscle tissues exposed to calcium (Calcified) were significantly increased (A) or decreased (B, C, D) compared to the control group (Normal) not exposed to calcium. * indicates p<0.05, and *** indicates p<0.001.

[0018] Figure 6 shows the results of confirming the efficacy of alleviating muscle differentiation inhibition by VIVIT peptide, an NFAT inhibitor. (A) is a graph showing the results of Western blotting to confirm Myh3 protein expression by VIVIT peptide in C2C12 myoblasts and its quantification, and (B) is a graph showing the results of Western blotting to confirm Myh3 and Pax7 protein expression by VIVIT peptide in mouse muscle-derived myoblasts and its quantification. *** indicates that Myh3 protein expression in the calcium-treated group significantly decreased and Pax7 expression significantly increased compared to the untreated control group, and p<0.001. ##, ### indicate that protein expression in the calcium and VIVIT peptide-treated groups significantly increased (Myh3) or decreased (Pax7) compared to the calcium-treated group, and ## indicates p<0.01, and ### indicates p<0.001. (C) is a graph showing the results of immunofluorescence analysis of Myh3 expression and subcellular localization in C2C12 myoblast cells, as well as its quantification. *** indicates that Myh3 expression in the calcium-treated group significantly decreased compared to the untreated control group, p<0.001, and ## indicates that Myh3 expression in the calcium and VIVIT peptide-treated group significantly increased compared to the calcium-treated group, p<0.01.

[0019] Figure 7 is a graph showing the results of confirming the inhibition effect of VIVIT peptide, an NFAT inhibitor, on the activation of the NFAT signaling system. The results are shown in the quantification graph of the expression level and subcellular localization of NFATc1-GFP by immunofluorescence analysis in C2C12 myoblast cells overexpressing NFATc1-GFP. *** indicates that the amount of nuclear influx of NFATc1 significantly increased in the calcium-treated group compared to the untreated control group, p<0.001, and ### indicates that the amount of nuclear influx of NFATc1 significantly decreased in the calcium and VIVIT peptide-treated group compared to the calcium-treated group, p<0.001.

[0020] The present invention relates to a pharmaceutical composition for preventing or treating muscle disease, comprising an NFAT inhibitor (Nuclear Factor Activated T cell inhibitor) as an active ingredient.

[0021] The above NFAT inhibitor is characterized by being (a) a peptide derived from an NFAT inhibitor, consisting of an amino acid sequence of SEQ ID NO: 1; or (b) a peptide derived from an amino acid sequence of SEQ ID NO: 1, wherein one or more amino acid residues are substituted, deleted, or inserted within the amino acid sequence of SEQ ID NO: 1, and which can alleviate inhibition of muscle differentiation.

[0022] In the above (a), the peptide derived from an NFAT inhibitor, consisting of the amino acid sequence of sequence number 1, is a 'VIVIT' peptide, which selectively inhibits the interaction between a protein belonging to the NFAT family and calcineurin without affecting the phosphatase activity of calcineurin.

[0023] A peptide derived from the amino acid sequence of SEQ ID NO: 1 may include a substitution, deletion or insertion of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1, and may have a function capable of alleviating inhibition of muscle differentiation, but is not limited thereto.

[0024] The above muscle disease is a muscle disease caused by muscle dysfunction, muscle atrophy, muscle wasting or muscle degeneration, and more preferably, it is any one selected from among atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, muscular dystrophy, amyotrophic lateral sclerosis, myasthenia gravis and sarcopenia, but is not limited thereto.

[0025] The pharmaceutical composition of the present invention may further include a carrier, excipient or diluent in addition to the NFAT inhibitor, and the pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is one commonly used in formulation, and includes, but is not limited to, saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, lactose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition to the above ingredients, it may further include antioxidants, buffers, bacteriostatic agents, diluents, surfactants, binders, lubricants, humectants, sweeteners, flavoring agents, emulsifiers, suspending agents, or preservatives.

[0026] The appropriate dosage of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. The pharmaceutical composition of the present invention for preventing or treating muscle diseases may be administered via any generally accepted route that can reach the target tissue.

[0027] The pharmaceutical composition of the present invention is not particularly limited thereto, but may be administered through a route such as oral administration, intramuscular administration, intraperitoneal administration, intravenous administration, subcutaneous administration, intradermal administration, transdermal patch administration, intranasal administration, intrapulmonary administration, or rectal administration, depending on the intended purpose, and preferably may be administered through a route of intramuscular administration or oral administration.

[0028] In addition, the present invention relates to a health functional food composition for promoting muscle growth, preventing or improving muscle loss or muscle strength loss, containing an NFAT inhibitor as an active ingredient.

[0029] The above health functional food composition is preferably manufactured in any one dosage form selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage, but is not limited thereto. The health functional food composition of the present invention can be manufactured by adding the NFAT inhibitor as it is or mixing it with other foods or food ingredients, and can be manufactured appropriately according to a conventional method. Examples of foods to which the NFAT inhibitor can be added include dairy products including caramel, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and includes all health functional foods in the conventional sense. That is, there is no particular limitation on the type of the above food. The above health functional food composition may contain various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavorings, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, it may contain fruit pulp for producing natural fruit juice and vegetable beverages. The above ingredients may be used independently or in combination. The health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, and the natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. The ratio of the above natural carbohydrates is not particularly important, but is preferably 0.01 to 0.04 g, more preferably 0.02 to 0.03 g, per 100 g of the composition of the present invention, but is not limited thereto.Sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartic acid.

[0030] In addition, the present invention relates to a veterinary composition for preventing or treating muscle disease, comprising an NFAT inhibitor as an active ingredient.

[0031] The veterinary composition of the present invention may further comprise suitable excipients and diluents according to conventional methods. Excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, ethanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil. The veterinary composition according to the present invention may further include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, etc., and the veterinary composition according to the present invention may be formulated using a method well known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to an animal, and the formulation may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile topical preparations, etc. The effective amount of the veterinary composition according to the present invention may be appropriately selected depending on the individual animal. It may be determined according to factors including the severity of the disease or condition, the sensitivity to the active ingredient of the present invention depending on the age, weight, health condition or sex of the individual, the route of administration, the period of administration, other compositions combined with or used simultaneously with the composition, and other factors well known in the physiological or veterinary fields.

[0032] In addition, the present invention relates to a feed additive for promoting muscle growth, preventing or improving muscle loss or muscle strength loss, containing an NFAT inhibitor as an active ingredient.

[0033] The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act. In the present invention, the term 'feed' may mean any natural or artificial diet, meal, etc., or a component of the meal, which is suitable for or intended for animals to eat, ingest, and digest. The type of the feed is not particularly limited, and feed commonly used in the relevant technical field may be used. Non-limiting examples of the feed include plant feeds such as grains, roots, fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils, starches, meal, or grain by-products; and animal feeds such as proteins, inorganic substances, oils, minerals, oils, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

[0034]

[0035] Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0036]

[0037] Materials and Methods

[0038] 1. NFAT inhibitor peptide

[0039] In the present invention, the effect of myogenic cell differentiation was evaluated using the peptide 'VIVIT', an NFAT inhibitor. Specifically, the VIVIT peptide has the function of selectively inhibiting the interaction between calcineurin and proteins belonging to the NFAT family without affecting the phosphatase activity of calcineurin.

[0040] The above VIVIT peptide is composed of an amino acid sequence of sequence number 1.

[0041] VIVIT peptide sequence information of the present invention peptidesequence sequence number VIVITMAGPHPVIVITGPHEE1

[0042]

[0043] 2. Culturing of mouse C2C12 myoblast cell line

[0044] Mouse C2C12 myoblasts were cultured in DMEM containing 10% FBS and 1% penicillin / streptomycin (Life Technologies Corp., Carlsbad, CA) at 37°C in a humidified incubator with 5% CO2. When the cell density reached 90%, differentiation was induced with DMEM containing 2% HS (horse serum) and 1% penicillin / streptomycin, and myofiber formation was confirmed after 5 days of differentiation.

[0045]

[0046] 3. Isolation of mouse muscle-derived myoblasts

[0047] To isolate myoblasts from 3-day-old C57BL / 6J mice, hind limb muscles were removed after anesthetizing the mice. The removed muscle tissue was washed with phosphate-buffered saline (PBS) and worked under aseptic conditions. The washed muscle tissue was finely chopped with small scissors and placed in DMEM medium containing 2 mg / mL collagenase (Collagenase Type II, Sigma-Aldrich) and 2.4 U / mL dispase II (Dispase II, Roche). Enzymatic digestion was performed for 1 hour in a humidified incubator at 37°C and 5% CO2. After the enzyme digestion was complete, the digest was filtered using a 70 μm cell strainer (Cell Strainer, Falcon). The filtered cell suspension was centrifuged at 1,000 rpm for 5 minutes to recover the cells, which were then allowed to adhere to a cell culture dish coated with poly-L-ornithine. After the cells were stably attached to the culture dish, the culture medium was replaced every 24 hours to remove non-adherent cells and residual tissue.

[0048]

[0049] 4. Culture and differentiation of mouse muscle-derived myoblasts

[0050] Isolated mouse muscle-derived myoblasts were cultured in a humidified incubator at 37°C and 5% CO2 in Ham's F-10 medium containing 10% bovine calf serum (BCS) and 1% penicillin / streptomycin. In addition, to induce differentiation, cells were seeded on cell culture plates coated with bovine skin-derived collagen, and when the cell density reached 90%, differentiation was induced with DMEM medium containing 2% horse serum (HS) and 1% penicillin / streptomycin, and the formation of myofibers was confirmed after 3 days of differentiation.

[0051]

[0052] 5. Human embryonic stem cell culture and differentiation

[0053] Human embryonic stem cells (WA09) were provided by WiCell Research Institute and cultured in a humidified incubator at 37°C and 5% CO2 using mTeSR™ medium (STEMCELL Technologies, USA) on cell culture plates coated with Matrigel®hESC-qualified matrix, LDEV-free (Corning, USA). To differentiate human embryonic stem cells, they were cultured for 1 week in Di-CL medium containing 1% ITS, 1% nonessential amino acids, 0.2% penicillin / streptomycin, 3 μM CHIR-99021, 0.5 μM LDN-193189, and 20 ng / mL recombinant FGF-2 in DMEM / F12 medium, and then cultured for 3 weeks in DK-HIFL medium containing 15% KSR, 1% nonessential amino acids, 0.2% penicillin / streptomycin, 0.1 mM 2-mercaptoethanol, 10 ng / mL recombinant HGF, 2 ng / mL recombinant IGF-1, 20 ng / mL recombinant FGF-2, and 0.5 μM LDN-193189 in DMEM / F12 medium. Embryonic stem cell-derived myoblasts were differentiated. Differentiated embryonic stem cell-derived myoblasts were cultured in SkGM™-2 (Skeletal Muscle Cell Growth Medium-2, Lonza). Differentiated embryonic stem cell-derived myoblasts were induced for muscle differentiation in DK-HI medium containing DMEM / F12 medium supplemented with 15% KSR, 1% nonessential amino acids, 0.2% penicillin / streptomycin, 0.1 mM 2-mercaptoethanol, 10 ng / mL recombinant HGF, and 2 ng / mL recombinant IGF-1. After 15 days of differentiation, myofiber formation was confirmed (Fig. 1A).

[0054]

[0055] Example 1. Immunofluorescence analysis of mouse C2C12 myoblast cell line

[0056] To analyze the expression level and subcellular localization of Myh3, a muscle differentiation marker protein, in C2C12 myoblasts continuously exposed to calcium, immunofluorescence analysis was performed. C2C12 cells treated with 3 mM and 6 mM calcium (CaCl2) for 5 days and the control group not treated with calcium were fixed with 4% paraformaldehyde solution and then permeabilized with 0.5% Triton X-100 in PBS. After that, nonspecific binding was blocked with 5% goat serum and 0.1% bovine serum albumin (BSA), and the expression level of Myh3 (Myosin heavy chain 3), a muscle differentiation marker gene, was analyzed using an antibody that binds to the Myh3 protein.

[0057] As a result, as disclosed in Figure 1A, the area of ​​myotube cells stained with Myh3 by calcium was significantly reduced. The Myh3 protein increases in expression level as myoblasts differentiate and forms myotubes. Through the decrease in the area of ​​myotube cells composed of Myh3 in myoblasts continuously exposed to calcium, it was confirmed that muscle differentiation of myoblasts is inhibited by continuous exposure to calcium.

[0058]

[0059] Example 2. Analysis of muscle differentiation marker gene expression in myoblasts

[0060] To analyze the expression levels of muscle differentiation marker genes in C2C12 myoblasts and mouse muscle-derived myoblasts continuously exposed to calcium, cell differentiation was induced under calcium-exposed conditions, and the protein and mRNA expression levels of muscle differentiation marker genes, Myh3 and MyoG (Myogenin), were analyzed. C2C12 myoblasts and mouse muscle-derived myoblasts were differentiated for 5 and 3 days, respectively, in differentiation medium supplemented with 3 mM and 6 mM calcium, and then proteins and RNA were extracted. Proteins were analyzed by Western blotting. The extracted proteins were electrophoresed on an acrylamide gel, and the separated proteins were transferred to a nitrocellulose film. The protein expression levels were confirmed using antibodies that specifically bind to Myh3 and MyoG, and the protein expression levels were compared with the expression levels of GAPDH. RNA was extracted using the Hybrid-R RNA extraction kit (GeneAll, Korea), and cDNA was synthesized using reverse transcriptase and random hexamers. The synthesized cDNA was subjected to RT-qPCR using the AriaMx real-time PCR system (Agilent Technologies, USA) to confirm mRNA expression levels. The mRNA expression levels were quantified by duplicate measurements, corrected with GAPDH, and presented in a graph.

[0061] As a result, the expression levels of Myh3 and MyoG were significantly reduced by calcium, and through these results, it was confirmed that muscle differentiation was inhibited in C2C12 myoblast cell lines and mouse muscle-derived myoblasts continuously exposed to calcium (Figures 1B, 1C).

[0062]

[0063] Example 3. Analysis of muscle differentiation marker gene expression in human embryonic stem cell-derived myoblasts.

[0064] To analyze the expression level of muscle differentiation marker proteins in human embryonic stem cell (WA09)-derived myoblasts continuously exposed to calcium, cell differentiation was induced under calcium-exposed conditions (Fig. 1D), and the protein expression level of MYH (Myosin heavy chain) was confirmed by Western blot.

[0065] As a result, as shown in Figure 1E, the protein expression level of MYH was significantly reduced by calcium.

[0066] Furthermore, the size of the formed myotubes was observed and measured, and as shown in Figure 1F, the size of the myotubes exposed to calcium decreased compared to the control group, and the area of ​​the myotubes was significantly reduced. These results confirmed that muscle differentiation of human embryonic stem cell-derived myoblasts was inhibited by continuous exposure to calcium.

[0067]

[0068] Example 4. Myoblast transcriptome analysis

[0069] To analyze the transcriptome of mouse muscle-derived myoblasts continuously exposed to calcium, RNA was extracted and sequenced. RNA from mouse muscle-derived myoblasts induced to differentiate in a differentiation medium containing 6 mM calcium for 3 days and from the control group was extracted using the Hybrid-R RNA Extraction Kit (GeneAll, Korea). After quality control and quantification of RNA using a Bioanalyzer 2100 (Agilent Technologies, USA), 1 μg of total RNA from each sample was selectively bound to a biotinylated probe using a kit (Illumina TruSeq Stranded Total RNA with Ribo-Zero, Illumina, USA) and ribosomal RNA (rRNA) was removed. cDNA was then synthesized using reverse transcriptase and random hexamers, and amplified by PCR to construct a cDNA library for sequencing. Finally, the library was quantified and quality-checked using the qPCR quantification protocol guide (KAPA Library Quantification Kits for Illumina Sequencing platforms) and DNA ScreenTape analysis (TapeStation D1000 ScreenTape, Agilent Technologies, USA), respectively. The generated cDNA library was sequenced using the Illumina HiSeq X Ten platform. The acquired transcriptome information was visualized using a volcano plot, and genes whose expression levels were significantly (p<0.05) more than twice as high under calcium-treated conditions compared to the control group were analyzed using gene ontology.

[0070] As a result, the expression levels of genes related to cell division and growth significantly increased in mouse muscle-derived myoblasts differentiated under conditions of continuous exposure to calcium, whereas the expression levels of genes related to muscle differentiation and muscle function significantly decreased. These results confirmed that when mouse muscle-derived myoblasts were differentiated under conditions of continuous exposure to calcium, myogenic differentiation of myoblasts was suppressed and the growth phase was maintained on the transcriptome (Fig. 2).

[0071]

[0072] Example 5. Analysis of myoblast growth induced by calcium

[0073] (1) Cell growth analysis

[0074] To analyze the cell growth of C2C12 myoblasts and mouse muscle-derived myoblasts continuously exposed to calcium, cell differentiation was induced under calcium-exposed conditions, and the cell nuclei were stained and counted. Mouse muscle-derived myoblasts and C2C12 myoblasts were induced to differentiate for 3 and 5 days, respectively, in differentiation medium supplemented with 3 mM and 6 mM calcium, fixed with 4% paraformaldehyde solution, and permeabilized with 0.5% Triton X-100 in PBS. Permeabilized cell nuclei were incubated for 15 minutes in PBS solution supplemented with 25 μg / mL Pi (Propidium iodide) and 12.5 μg / mL RNase A, and the cell nuclei were stained.

[0075] As a result, the number of cell nuclei in C2C12 myoblasts and mouse muscle-derived myoblasts continuously exposed to calcium significantly increased despite the differentiation-inducing conditions. These results confirmed that both types of myoblasts did not differentiate upon calcium exposure and maintained a growth phase (Figs. 3A, 3B).

[0076]

[0077] (2) Analysis of growth indicator gene expression levels

[0078] We investigated the protein and mRNA expression levels of the Pax7 (Paired box protein), a growth indicator of mouse muscle-derived myoblasts continuously exposed to calcium. Western blot and RT-qPCR were performed on protein and RNA extracted from mouse muscle-derived myoblasts differentiated for 3 days in a differentiation medium supplemented with 3 mM and 6 mM calcium, as well as from the control group. The expression level of Pax7 mRNA was quantified by duplicate measurements, corrected with GAPDH, and presented in a graph.

[0079] As a result, as disclosed in Figure 3C, the protein and mRNA expression levels of Pax7 were significantly increased by calcium. While the expression level of Pax7 significantly decreased during the differentiation stage, the expression level of Pax7 increased in myoblasts continuously exposed to calcium, confirming that mouse muscle-derived myoblasts were maintained in the growth stage rather than the differentiation stage by calcium.

[0080]

[0081] Example 6. Analysis of NFAT signaling pathway activation in myoblasts

[0082] (1) Analysis of signal transmission system activation through GSEA analysis

[0083] The activation of signaling pathways in mouse muscle-derived myoblasts exposed to continuous calcium was analyzed using the GSEA (Gene Set Enrichment Analysis) method. Based on transcriptome RNA sequencing results from mouse muscle-derived myoblasts exposed to continuous calcium, GSEA analysis was performed using GSEA software version 2.2.2.0 against gene sets in MSigDB v5.0. GSEA analysis was performed on the following genes: NFATc1 signaling pathway, a downstream signaling pathway of calcineurin, a calcium-dependent signaling pathway; calcineurin signaling pathway activated by intracellular calcium; DNA replication, a marker of cell division; and myogenesis-related gene sets.

[0084] As a result, as shown in Fig. 4A, it was confirmed that the calcineurin and NFATc1 signaling systems were activated in mouse muscle-derived myoblasts continuously exposed to calcium, whereas the muscle differentiation-related systems were inactivated.

[0085]

[0086] (2) Analysis of NFAT signaling pathway activation

[0087] We investigated the mRNA expression of Ccnd1 (Cyclin D1), a growth indicator gene for myoblasts whose expression is induced by the NFAT signaling pathway. Mouse muscle-derived myoblasts and the C2C12 myoblast cell line were differentiated for 3 and 5 days, respectively, in a differentiation medium supplemented with 6 mM calcium. RNA was extracted, cDNA was synthesized, and RT-qPCR was performed. The expression level of Ccnd1 mRNA was quantified by duplicate measurements, corrected with GAPDH, and presented in a graph.

[0088] As a result, as shown in Fig. 4B, the expression level of Ccnd1 mRNA was significantly increased in C2C12 myoblast cell lines and mouse muscle-derived myoblasts continuously exposed to calcium, confirming that the NFAT signaling system of mouse muscle-derived myoblasts was activated by calcium.

[0089]

[0090] Example 7. Analysis of muscle tissue derived from patients with calcific tendonitis

[0091] To investigate the growth of myoblasts and the activation of the NFAT signaling pathway in muscle tissue directly exposed to calcium crystals, myoblasts were isolated from the tissues of patients with calcific tendinitis in which calcium crystals form in the supraspinatus muscle. Supraspinatus muscles were surgically isolated from eight patients with calcific tendinitis, and RNA was extracted from tissues directly exposed to calcium and tissues not in close proximity to calcium. The mRNA expression levels of CCND1 (Cyclin D1), a growth indicator gene and NFAT signaling pathway target gene, MYH3 (Myosin Heavy Chain 3), a muscle differentiation indicator gene, and SPRY1 (Sprouty RTK Signaling Antagonist 1) and PAX3 (paired box gene 3), which are myoblast resting marker genes, were determined by RT-qPCR. The mRNA expression levels were quantified in duplicate, corrected for GAPDH, and presented in a graph.

[0092] As a result, the expression of CCND1, a target gene of the NFAT signaling pathway, significantly increased in myoblasts in muscle tissue directly exposed to calcium crystals, whereas the expression of MYH3, a muscle differentiation marker gene, and SPRY1 and PAX3, which are resting marker genes, significantly decreased. These results confirmed that myoblasts in muscle tissue directly exposed to calcium crystals did not enter the resting or differentiation phase, but maintained the growth phase with suppressed muscle differentiation (Fig. 5).

[0093]

[0094] Example 8. Analysis of the efficacy of NFAT inhibitor (VIVIT peptide) in alleviating muscle differentiation inhibition.

[0095] (1) Analysis of the efficacy of NFAT inhibitors to alleviate muscle differentiation marker protein inhibition

[0096] We confirmed the efficacy of VIVIT peptide, which suppresses the activation of the intracellular NFAT signaling pathway, in alleviating the inhibition of muscle differentiation. Differentiation was induced in mouse muscle-derived myoblasts and C2C12 myoblasts for 3 and 5 days, respectively, in a differentiation medium supplemented with 6 mM calcium and 20 μM VIVIT peptide. Proteins were isolated and subjected to Western blotting. Protein expression levels were corrected with GAPDH and presented graphically.

[0097] As a result, while the Myh3 protein expression level of C2C12 myoblast cell line and mouse muscle-derived myoblast cells was decreased by calcium, the Myh3 protein expression level was significantly increased when treated with the VIVIT peptide of the present invention. While the Pax7 protein expression level of mouse muscle-derived myoblast cells was increased by calcium, the Pax7 protein expression level was significantly decreased when treated with the VIVIT peptide of the present invention (Figs. 6A, 6B).

[0098] In addition, when the expression level of Myh3 was analyzed in C2C12 myoblast cell lines by immunofluorescence analysis using an antibody that binds to the Myh3 protein, as shown in Fig. 6C, it was confirmed that the area of ​​myotube cells stained with Myh3 by calcium significantly decreased, whereas when the VIVIT peptide of the present invention was treated, the area of ​​myotube cells composed of Myh3 significantly increased.

[0099] Considering that Myh3 protein expression level increases as myoblasts differentiate and constitute myotube cells, and Pax7 protein expression level decreases during the differentiation stage, we confirmed that VIVIT peptide effectively inhibits the activation of the NFAT signaling pathway in C2C12 myoblast cell line and mouse muscle-derived myoblasts continuously exposed to calcium, thereby alleviating the inhibition of muscle differentiation.

[0100]

[0101] (2) Analysis of the efficacy of NFAT inhibitors to alleviate the activation of the NFAT signaling system

[0102] To confirm that the effect of VIVIT peptide in alleviating muscle differentiation inhibition in myoblasts exposed to excessive calcium was due to its inhibitory effect on NFAT signaling pathway activation, we verified the effect of VIVIT peptide in inhibiting NFAT signaling pathway activation in treated myoblasts. After overexpressing NFATc1-GFP in C2C12 myoblasts, differentiation was induced for 5 days in a differentiation medium supplemented with 6 mM CaCl2 and 20 μM VIVIT peptide. The activation of NFAT signaling pathway was analyzed based on the subcellular localization of the overexpressed NFATc1-GFP by immunofluorescence analysis using an antibody that binds to GFP protein.

[0103] As a result, as shown in Fig. 7, the amount of NFATc1-GFP that was introduced from the cytoplasm into the nucleus was significantly increased in myoblasts in which muscle differentiation was inhibited due to exposure to excessive calcium, indicating that the NFAT signaling system of myoblasts was activated by excessive calcium exposure. On the other hand, when the VIVIT peptide was treated to myoblasts exposed to calcium, the nuclear influx of NFATc1-GFP in myoblasts was significantly inhibited, confirming that the VIVIT peptide inhibits the activation of the NFAT signaling system of myoblasts.

[0104] Through these results, it was confirmed that muscle differentiation was inhibited when the NFAT signaling system of myoblasts was activated by excessive calcium exposure, whereas the NFAT signaling system activation was inhibited by treatment with the VIVIT peptide, an NFAT inhibitor of the present invention, thereby alleviating the inhibition of muscle differentiation.

Claims

1. A pharmaceutical composition for preventing or treating muscle disease, comprising an NFAT inhibitor (Nuclear Factor Activated T cell inhibitor) as an active ingredient.

2. A pharmaceutical composition for preventing or treating muscle disease, characterized in that the NFAT inhibitor in claim 1 is (a) a peptide derived from an NFAT inhibitor, consisting of an amino acid sequence of SEQ ID NO: 1; or (b) a peptide derived from an amino acid sequence of SEQ ID NO: 1, wherein one or more amino acid residues are substituted, deleted, or inserted within the amino acid sequence of SEQ ID NO: 1, and which can alleviate inhibition of muscle differentiation.

3. A pharmaceutical composition for preventing or treating muscle disease, characterized in that the muscle disease in claim 1 is any one selected from the group consisting of atony, muscular atrophy, muscular dystrophy, myasthenia gravis, rigid spine syndrome, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and sarcopenia.

4. A pharmaceutical composition for preventing or treating muscle disease, characterized in that, in addition to the NFAT inhibitor in paragraph 1, it further comprises a carrier, excipient or diluent.

5. A health functional food composition containing an NFAT inhibitor as an active ingredient for promoting muscle growth and preventing or improving muscle loss or muscle strength loss.

6. A health functional food composition for promoting muscle growth, preventing or improving muscle loss or muscle strength loss, characterized in that the NFAT inhibitor in paragraph 5 is manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, and beverages.

7. A veterinary composition for the prevention or treatment of muscle disease, comprising an NFAT inhibitor as an active ingredient.

8. Feed additive for promoting muscle growth, preventing or improving muscle loss or muscle strength loss, containing an NFAT inhibitor as an active ingredient.

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