Treatments for muscle atrophy and screening methods

Abstract

The objective is to provide muscle atrophy inhibitors and muscle strengthening agents that are effective for muscle atrophy resulting from various causes, such as myogenic muscle atrophy, neurogenic muscle atrophy due to motor nerve damage, and disuse muscle atrophy due to decreased activity such as prolonged bed rest, as well as for strengthening the muscles of livestock. [Solution] We have revealed that Mkl1 / 2 directly binds to the FoxO protein, which controls the gene cluster that causes muscle atrophy, and inhibits its expression. Furthermore, we have revealed that Mkl1 / 2 also inhibits the transcriptional activity of glucocorticoid receptors. Introduction of Mkl1 / 2 via a viral vector resulted in increased muscle mass and suppression of the expression of the gene cluster that causes muscle atrophy. By using substances that enhance the expression of Mkl1 / 2 or substances that inhibit its degradation, muscle atrophy can be suppressed and muscle can be strengthened. In addition, since we have found a new target molecule effective against muscle atrophy, compounds can be screened using this as an indicator.

Description

Technical Field

[0001] The present invention relates to a therapeutic agent for muscle atrophy and a muscle strengthening agent. The present invention also relates to a method for screening these pharmaceutical compositions.

Background Art

[0002] Muscle atrophy, which involves muscle loss, arises from various causes. Known causes include myogenic muscle atrophy caused by diseases of the muscle itself, neurogenic muscle atrophy caused by motor nerve disorders, and disuse muscle atrophy caused by decreased activity such as long-term bedridden status. For example, sarcopenia refers to a condition in which muscle mass decreases with aging, and with the increase in the elderly population, the proportion of people diagnosed with primary sarcopenia caused solely by aging is increasing. In general, muscle mass gradually decreases starting from around 40 years of age. At 70 years of age, compared with people in their 20s, skeletal muscle area decreases by 25 to 30%, muscle strength decreases by 30 to 40%, and affected people experience subjective symptoms such as difficulty carrying heavy loads. In addition to age-related sarcopenia, secondary sarcopenia can also occur due to nutritional deficiency, diseases and other factors. Disuse atrophy caused by disuse of muscles such as prolonged bedridden status is also increasing along with the growth of the elderly population. It is known that muscle atrophy is caused by various factors, including drugs such as steroids, endocrine diseases, cancer, wasting diseases such as hyperthyroidism and diseases accompanied by high fever, chronic kidney disease, chronic liver disease, chronic heart failure, and mental disorders such as depression. Since muscle atrophy can arise from various causes, its pathogenesis remains unclear. Therefore, development of drugs for treating muscle diseases is desired.

[0003] Furthermore, in the livestock industry, increasing muscle mass leads to increased profits, so enhancing muscle mass is an important issue along with meat quality. Currently, optimal fattening conditions for each animal species are studied through examining breeding conditions such as improved feed and feeding methods. However, due to the existence of various breeding conditions, it is difficult to establish optimal breeding conditions. Attempts to increase muscle mass through genetic modification have also been studied, but due to concerns over genetically modified animals, it is considered difficult to be widely popularized among the general public.

[0004] Regarding gene expression involved in sarcopenia, muscle atrophy, or muscle mass enhancement, the following reports have already been made. With aging, the quadriceps femoris muscle exhibits aging phenotypes such as a decrease in muscle cross-sectional area, but it has been reported that the expression of SRF, MRTF-A(Mkl1), and STARS genes decreases, while the expression of RhoA and RhoGD1 genes increases in the quadriceps femoris and triceps brachii muscles (Non-Patent Literature 1). Although this document describes age-related muscle atrophy and the associated changes in gene expression, the details of the signaling pathway are not clarified, and the genes that control muscle atrophy remain unknown. Patent Literature 1 presents the results of comprehensive gene analysis of skeletal muscle in immobilization-induced muscle atrophy model mice and patients with immobilization-induced muscle atrophy, which found that the expression of the chemokine CXCL10 is enhanced by immobilization, and suggests the possibility that muscle atrophy can be suppressed by inhibiting its expression. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2022-162858 [Non-patent literature]

[0006] [Non-Patent Document 1] Sakuma et al., Biochimica et Biophysica Acta,2008, Vol.1782,pp.453-461 [Non-Patent Document 2] Milan, G. et al., Nature Communications, 2015, DOI: 10.1038 / ncomms7670 [Non-Patent Document 3] Li, J., et al., Proc Natl Acad Sci U SA,, 2005. Vol.102(25): p. 8916-8921. [Non-Patent Document 4] Oh, J., et al., Proc Natl Acad Sci USA, 2005. Vol. 102(42): p. 15122-15127. [Overview of the project] [Problems that the invention aims to solve]

[0007] While the above-mentioned literature presents findings obtained from gene analysis that increases or decreases during muscle atrophy, it does not clarify the mechanism of muscle atrophy onset. Therefore, it is unlikely that it can suppress muscle atrophy caused by various factors. Furthermore, no effective muscle atrophy inhibitors have been developed to date, and their development is highly desired.

[0008] The inventors created Mkl2 gene knockout mice and found a phenotype characterized by a significant reduction in white adipose tissue. Therefore, they analyzed the mechanism of gene regulation by Mkl2 and discovered that it suppresses the transcription factors Foxo1, Foxo3, and Foxo4, which belong to the FoxO subfamily. FoxO transcription factors are known to activate atrogenes (genes that cause muscle atrophy) and induce muscle atrophy during various catabolic processes in skeletal muscle. Therefore, they analyzed the actions of Mkl1 and Mkl2 in skeletal muscle and found that they regulate the expression of atrogenes, thus completing the present invention. [Means for solving the problem]

[0009] The present invention relates to the following pharmaceutical compositions and screening methods. (1) A muscle atrophy inhibitor and / or muscle enhancer characterized by having Mkl1 and / or Mkl2 as target molecules and containing as an active ingredient a substance that increases the amount of Mkl1 and / or Mkl2. The inventors of the present invention have discovered that Mkl1 and Mkl2 regulate Foxo1, Foxo3, Foxo4, and the glucocorticoid receptor (GR), which are involved in inducing the expression of muscle atrophy-causing genes, and have completed the present invention.

[0010] (2) The muscle atrophy inhibitor and / or muscle enhancer according to (1), characterized by increasing the amount of Mkl1 and / or Mkl2 in the nucleus. We discovered that Mkl1 / 2 directly binds to Foxo1 / 3 / 4. Therefore, by increasing the amount of Mkl1 / 2 in the nucleus, we can suppress the transcriptional activity of Foxo1 / 3 / 4, thereby inhibiting muscle atrophy and enhancing muscle mass.

[0011] (3) The muscle atrophy inhibitor and / or muscle strengthening agent according to (1), characterized in that the muscle atrophy inhibitor is used for the treatment of muscle atrophy caused by aging, disuse, neurogenic, malnutrition, or steroid administration. We found that increasing Mkl1 / 2 expression has an effect on muscle atrophy caused by various factors. Therefore, it can be expected to suppress muscle atrophy and strengthen muscles regardless of the cause.

[0012] (4) The muscle atrophy inhibitor and / or muscle enhancer according to (1), characterized by introducing Mkl1 and / or Mkl2 using a viral vector. Mkl1 was introduced into mice using adeno-associated virus, resulting in enhancement of skeletal and cardiac muscle. Therefore, viral vector therapy is feasible.

[0013] (5) The muscle atrophy inhibitor and / or muscle enhancer according to (1), characterized in that it increases the amount of Mkl1 and / or Mkl2 by suppressing the degradation of Mkl1 and / or Mkl2. Ubiquitin-proteasome inhibitors suppressed the degradation of Mkl1 / 2, resulting in stable expression. Therefore, suppressing the degradation of Mkl1 / 2 can also suppress muscle atrophy.

[0014] (6) A muscle atrophy inhibitor and / or muscle enhancer according to any one of (1) to (5), characterized in that the muscle is skeletal muscle or cardiac muscle. In experiments using mice, an increase in the weight of skeletal muscle and cardiac muscle was observed.

[0015] (7) A method for screening a muscular atrophy inhibitor and / or a muscle strengthening agent, which comprises exposing a candidate compound to cultured cells, and using an increase in the amount of Mkl1 and / or Mkl2 in the cultured cells as an indicator. The present inventors have shown that the expression regulation of muscle atrophy-causing gene groups by glucocorticoid receptors and the transcription factors Foxo1 / 3 / 4 is controlled by Mkl1 / 2. Therefore, a muscular atrophy inhibitor and a muscle strengthening agent can be screened using the expression of Mkl1 or Mkl2 as an indicator.

[0016] (8) The method for screening a muscular atrophy inhibitor and / or a muscle strengthening agent according to (7), wherein a reporter protein is fused to Mkl1 and / or Mkl2 protein, and an increase in the reporter protein is used as the indicator. Use of a system that monitors Mkl1 and Mkl2 expression via a reporter protein is also compatible with high-throughput screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] [Figure 1A] A figure showing the results of microarray analysis of mRNA expression in white adipose tissue of wild-type mice and Mkl2 knockout mice. [Figure 1B] A figure showing genes with high mRNA expression in white adipose tissue of Mkl2 knockout mice. [Figure 2] A figure showing that skeletal muscle weight is reduced in Mkl2 knockout mice. [Figure 3A] A figure showing the results of analysis of Mkl1 and Mkl2 expression in gastrocnemius muscle after 4 weeks of forced treadmill exercise in C57BL / 6J mice. [Figure 3B] A figure showing the results of western blotting analysis of the intracellular localization of Mkl2 in gastrocnemius muscle immediately after forced treadmill exercise in C57BL / 6J mice. [Figure 3C]Microscopic image showing the results of analyzing the intracellular localization of Mkl1-eGFP and Mkl2-eGFP expressed in myotubes derived from C2C12 cells. [Figure 3D] Microscopic images showing the migration of Mkl2 from the cytoplasm to the nucleus due to electrical stimulation-induced myotubular contraction. The two images on the right show high-magnification of the area enclosed by the square in the image on the left. [Figure 4A] This figure shows the results of gene expression analysis of the Mkl1 and Mkl2 genes, as well as the gene groups responsible for muscle atrophy, in the gastrocnemius muscle of young (8 weeks old) and old (119 weeks old) mice. [Figure 4B] A figure showing the correlation between the expression levels of Mkl1 and Atrogin1, MuRF1, Foxo1, and Foxo3 in the gastrocnemius muscle of mice at different ages. [Figure 5] This figure shows the results of an analysis of changes in the expression of muscle atrophy-causing genes and the Mkl1 and Mkl2 genes in the anterior tibialis muscle of mice that underwent sciatic nerve resection of the right hind limb. [Figure 6] This figure shows the results of an analysis of the gene groups responsible for muscle atrophy due to fasting, their regulatory factors (Foxo1 / 3 / 4), and changes in the expression of the Mkl1 and Mkl2 genes. [Figure 7A] This figure shows the results of an analysis of the effects of Mkl1 and Mkl2 expression on the expression of gene groups causing muscle atrophy, and Foxo3, induced by steroid addition to myotubes derived from C2C12 cells. [Figure 7B] This figure shows the results of an analysis of the effects of Mkl1 and Mkl2 on the transcriptional activity of the glucocorticoid receptor (GR) in a luciferase assay using glucocorticoid receptor response elements. [Figure 8A] This figure shows the results of an analysis of the effects of Mkl1 (top panel) and Mkl2 (bottom panel) on the transcriptional activity of Foxo1, Foxo3, and Foxo4 in a luciferase assay using Foxo response elements. [Figure 8B] This figure shows the results of an analysis of the effects of Mkl1 (top panel) and Mkl2 (bottom panel) on the transcriptional activity of Foxo1, Foxo3, and Foxo4 in a luciferase assay with a FoxO response element in 293FT cells, in the presence of the PI3K inhibitor LY294002. [Figure 8C] This figure shows the results of analyzing the expression of muscle atrophy-causing genes in myotubes derived from C2C12 cells, where Foxo1, Mkl1, and Mkl2 were overexpressed. The left side shows the mRNA expression analysis, and the right side shows the protein expression analysis. [Figure 8D] This figure shows the results of analyzing the expression of muscle atrophy-causing genes in myotubes derived from C2C12 cells, where Foxo3, Mkl1, and Mkl2 were overexpressed. The left side shows the mRNA expression analysis, and the right side shows the protein expression analysis. [Figure 8E] This figure shows the results of analyzing the expression of muscle atrophy-causing genes in myotubes derived from C2C12 cells after adding the PI3K inhibitor LY294002 and the Mkl1 / 2 nuclear localization inhibitor CCG-1423. The left side shows the mRNA expression analysis, and the right side shows the protein expression analysis. [Figure 8F] This figure shows the results of analyzing the expression of muscle atrophy-causing genes after suppressing the expression of Mkl1 or Mkl2 using artificial microRNA (miRNA) in myotubes derived from C2C12 cells. The upper left panel schematically shows the structure of the designed miRNA. The upper right panel shows the results confirming that the expression of Mkl1 and Mkl2 was suppressed by the miRNA. The lower panel shows the results of analyzing mRNA expression (left) and protein expression (right) to confirm that the expression of muscle atrophy-causing genes increased as a result of suppressing Mkl1 and Mkl2 expression by miRNA. [Figure 8G] This figure shows the inhibitory effect of Mkl1 or Mkl2 on muscle atrophy induced by expressing Foxo1 or Foxo3 in C2C12-derived myotubes. [Figure 9A] This figure shows the results of analyzing the binding of Mkl1 and Mkl2 to Foxo1 using immunoprecipitation. [Figure 9B] This figure shows the results of analyzing the binding of Mkl1 and Mkl2 to Foxo1 using a split luciferase assay. [Figure 9C] This figure shows the results of analyzing the binding of Mkl1 to Foxo3 and Foxo4, and the binding of Mkl2 to Foxo3 and Foxo4, using a split luciferase assay. [Figure 9D]This figure shows the results of a split ciferase assay analyzing the binding of Foxo1 to Mkl1 or Mkl2 in the presence of CCG-1423, a Mkl1 / 2 nuclear localization inhibitor. [Figure 10A] This figure shows the results of analyzing the region necessary for Foxo1 and Mkl2 binding by creating deletion mutants starting from the N-terminus. [Figure 10B] This figure shows that the transcriptional activation domain (TAD) of Mkl2 acts on the full-length Foxo1 protein (left) and the Foxo1 protein DNA-binding domain (Forkhead domain), suppressing transcriptional activity. [Figure 10C] This figure shows the results of analyzing changes in Foxo3 binding to genomic regions surrounding muscle atrophy-causing genes, induced by Mkl1 or Mkl2 expression, using chromatin immunoprecipitation (ChIP) assay. [Figure 11A] This paper presents the results of an analysis of the time-dependent changes in Akaluc expression in mice systemically administered with adeno-associated virus (MyoAAV.2A Mhck7-Akaluc). [Figure 11B] This figure shows the expression of eGFP in various muscles and the heart of mice that received systemic administration of adeno-associated virus (MyoAAV.2A Mhck7-eGFP). [Figure 11C] This figure shows the results of a comparative analysis of Foxo1 / 3 / 4 expression induction under fasting conditions, after expressing Mkl1 in the muscle of C57BL / 6J mice using adeno-associated virus. [Figure 11D] This figure shows the results of analyzing the expression of muscle atrophy-causing genes and Foxo1 / 3 / 4 when Mkl1 was introduced into the muscle of C57BL / 6J mice using adeno-associated virus and dexamethasone sodium phosphate was administered intraperitoneally. [Figure 11E] This figure shows the results of analyzing the muscle weight of the gastrocnemius muscle after systemic administration of adeno-associated virus (MyoAAV.2A Mhck7-Mkl1) to mice. [Figure 11F] This figure shows the results of analyzing the weight of the heart after systemic administration of adeno-associated virus (MyoAAV.2A Mhck7-Mkl1) to mice. [Figure 12A]This figure shows the results of analyzing the half-lives of Mkl1 and Mkl2 after expressing them using the Tet-On promoter and removing doxycycline, which is an expression inducer. [Figure 12B] This figure shows the results of analyzing the stability of Mkl1 and Mkl2 proteins using ubiquitin-proteasome inhibitors. [Figure 13] A schematic diagram illustrating the points of action of Mkl1 and Mkl2 in muscle atrophy and muscle mass increase. [Modes for carrying out the invention]

[0018] In this specification, a therapeutic agent for muscle atrophy or a muscle-enhancing agent can be any substance that can increase the amount of Mkl1 or Mkl2 in the muscle. For example, it may be a substance that enhances Mkl1 or Mkl2 expression, or a substance that increases the amount in the cell by inhibiting its degradation. It may also include any form of substance. Specifically, it includes small molecule compounds, nucleic acid drugs, peptides and protein preparations, and biological products introduced into the body by viral vectors such as adenovirus vectors and adeno-associated virus vectors. Furthermore, muscle-enhancing agents administered to livestock are preferably substances extracted from natural materials such as plants, since livestock are fattened for meat production.

[0019] The method of administering the pharmaceutical composition of the present invention to a patient or animal can be appropriately determined depending on the substance of the active ingredient, but it may be administered orally or parenterally. If administered orally, preferred dosage forms include solid preparations (including tablets, capsules, drops, and lozenges), granules, powders, and liquid preparations (including internal liquid preparations, emulsions, and syrups). If a solid preparation is used, it may be a dosage form with a coating known in the art, such as a sugar-coated tablet, a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, or a multi-layer tablet, as needed. Furthermore, if administered to livestock, it may be in a form that can be mixed with feed.

[0020] For parenteral administration, subcutaneous, intramuscular, intravenous, intraperitoneal, or intranasal administration is useful. Preferred embodiments include subcutaneous or intramuscular injection. Other preferred embodiments include intravenous drip infusion or intravenous injection. The pharmaceutical composition may also contain pharmaceutically acceptable carriers. Specific pharmaceutically acceptable carriers include all physiologically acceptable solvents, dispersions, antibacterial or antifungal agents, isotonic agents, and absorption retarders. Examples of pharmaceutically acceptable carriers include water, physiological saline, phosphate-buffered saline, dextrose, glycerin, and ethanol, and the composition may contain at least one of these carriers. Pharmaceutically acceptable carriers may also contain small amounts of substances that enhance efficacy, such as humectants, emulsifiers, preservatives, and buffers. Regarding the administration interval, it is desirable to continue treatment for an appropriate period while monitoring the therapeutic effect.

[0021] Furthermore, the pharmaceutical composition of the present invention can be administered continuously from the perspective of preventing muscle atrophy, such as for the prevention of sarcopenia or as a preventative measure against illness. The dosage may be lower than that used for therapeutic purposes. From the perspective of preventing illness, it is not necessary to actively increase Mkl1 and Mkl2, and it may suffice to suppress the decrease in their expression. In addition, it may be continuously taken in the form of a supplement or nutritional supplement rather than as a pharmaceutical composition.

[0022] Muscle atrophy is known to be caused by the activation of multiple genes known as the muscle atrophy-causing genes. Furthermore, the expression of the muscle atrophy-causing genes is induced by the nuclear receptor glucocorticoid receptor (GR) and the transcription factors Foxo1, Foxo3, and Foxo4, and it has been reported that mice lacking Foxo1 / 3 / 4 in muscle tissue show resistance to muscle atrophy (Non-Patent Literature 2). The FoxO protein is a transcription factor belonging to the Forkhead-type transcription factor family, and it is known that there are four isoforms in mammals: Foxo1, Foxo3, Foxo4, and Foxo6. The inventors of this invention created Mkl2 knockout mice and analyzed their phenotype, finding that white adipose tissue was significantly reduced. Furthermore, in the process of analyzing the molecular mechanism, we discovered that Mkl1 and Mkl2 suppress the transcriptional activation ability of transcription factors Foxo1, Foxo3, and Foxo4, and further suppress the transcriptional activation ability of glucocorticoid receptors responsible for inducing Foxo expression, and as a result suppress the expression of muscle atrophy-causing genes, thus completing the present invention (see Figure 13).

[0023] The following explanation will be accompanied by data. Unless otherwise noted, the following analyses were performed using methods commonly employed in this field. Mkl2 is known to be a coactivator of the transcription factor Srf, but Mkl2 knockout mice (hereinafter referred to as Mkl2 KO mice, or simply Mkl2 KO) have been reported to be embryonically lethal due to cardiovascular developmental abnormalities such as hypoplasia of the sixth pharyngeal arch artery and defects of the interventricular septum (Non-Patent Literature 3, 4). Therefore, the function of Mkl2 from late development to adulthood was unknown. The inventors have found that heterosis by crossing ICR background with C57BL / 6N background can avoid embryonic lethality and produce Mkl2 KO mice.

[0024] Specifically, Mkl2 KO mice were created as follows: Mkl2 KO mice were converted from KOMP to ES cells (Mrtfb TM1a(KOMP)Mbp I purchased and bred (Mrtfb).TM1a(KOMP)Mbp Mice were sequentially crossed with CAG-FRT mice and CAG-Cre mice to generate heterozygous mice lacking exons 11 and 12. These heterozygous mice were maintained in the C57BL / 6N mouse strain and the ICR mouse strain, and hybrid vigor between the two strains generated Mkl2 KO mice lacking both alleles.

[0025] Although the role of Mkl1, a paralog gene of Mkl2, in adipogenesis has already been reported, its function is not clearly understood. Currently, there are conflicting reports: some suggest that Mkl1 negatively regulates white adipocyte differentiation, while others suggest that Mkl1 differentiates mesenchymal stem cells into white adipocytes but not into brown adipocytes. Therefore, to investigate the mechanism of white adipose tissue reduction in Mkl2 KO mice and the effects of Mkl2 deficiency on various transcription factors, RNA was extracted from the inguinal white adipose tissue of Mkl2 KO mice and their littermates, and their expression was analyzed (Figure 1).

[0026] RNA extracted from adipose tissue was analyzed for expression using microarray analysis (Figure 1A). Mkl2 KO mice and wild-type littermates were euthanized, and white adipose tissue was isolated from the groin. TRIzol Reagent (Thermo Fisher Scientific) was added, and the tissue was disrupted using Micro Smash MS-100R. RNA purification was performed according to the reagent manual. The purified RNA was subjected to microarray analysis using 3D-Gene (TORAY). Genes showing differences in expression between wild-type and Mkl2 KO mice were extracted. Genes showing altered expression in Mkl2 KO mice were extracted (Figure 1B), and further, upstream regulatory genes that could explain these gene expression changes were analyzed using Ingenuity™ Pathway Analysis (IPA). Changes in the activation / inactivation state of transcription factors in the white adipose tissue of Mkl2 KO mice were predicted, and the top 10 transcription factors located upstream of RNA showing altered expression by microarray analysis were extracted (Table 1).

[0027] [Table 1]

[0028] IPA analysis predicted that the transcriptional activity of the transcription factor Srf, which is known to interact with Mkl2 and for which Mkl2 functions as a coactivator, was suppressed. Furthermore, it was estimated that Foxo1 was activated higher in the list than Srf. By analyzing the phenotype of reduced white adipose tissue, the inventors found that Mkl1 and Mkl2 suppress the transcription factor Foxo1. As mentioned above, Foxo1 / 3 / 4 transcription factors are known to activate muscle atrophy-causing genes and induce muscle atrophy in various catabolic processes of skeletal muscle. Therefore, the involvement of Mkl2 and its paralog, Mkl1, in muscle atrophy was analyzed.

[0029] In Mkl2 KO mice, the reduction in skeletal muscle weight was measured (Figure 2). The gastrocnemius muscle, soleus muscle, plantaris muscle, tibialis anterior muscle (TA), and extensor digitorum longus muscle (EDL) were extracted from 22-week-old Mkl2 KO mice, wild-type (WT), and heterozygous (Ht) mice, and their muscle weights were measured. All muscle weights were significantly reduced in Mkl2 KO mice compared to wild-type and heterozygous mice. Therefore, it was revealed that Mkl2 KO significantly reduces skeletal muscle weight. In this specification, * indicates P<0.05 and ** indicates P<0.01.

[0030] Therefore, we analyzed the behavior of Mkl1 and Mkl2 in muscle tissue. Ten-week-old C57BL / 6J mice were subjected to forced exercise on a treadmill for 60 minutes / day for four weeks. The treadmill was started at a speed of 15 m / min, and the speed was increased by 1 m / min every 10 minutes until it reached 20 m / min. After four weeks of forced exercise, the expression of Mkl1 and Mkl2 in the gastrocnemius muscle was analyzed by RT-qPCR (n=3). In the exercise group (Exc; Exercise), the expression of Mkl1 and Mkl2 was increased by approximately 2.5 times compared to the non-exercise group (Sed; Sedentary) (Figure 3A). In addition, the intracellular localization of Mkl2 protein in the gastrocnemius muscle of mice in the treadmill exercise group and the non-exercise group was analyzed by Western blotting. The cytoplasm and nuclei of gastrocnemius muscle tissue, immediately after 3 hours of treadmill exercise, were fractionated using the Subcellular Protein Fractionation Kit for Tissues (Thermo Fisher Scientific) and analyzed by Western blotting. MKL2 / MRTF-B antibody (Cell Signaling Technology) was used as the primary antibody, and HRP-labeled anti-rabbit IgG (Cell Signaling Technology) was used as the secondary antibody. Chemiluminescence (Chemi-Lumi One Super, Nacalai Tesque) was used for measurement. Comparing the amounts of Mkl2 protein in the exercise and non-exercise groups, there was no change in Mkl2 levels in the cytoplasmic fraction between the two groups, but a significant increase in Mkl2 levels in the nuclear fraction was observed after exercise. This revealed that Mkl2 translocates to the nucleus during exercise (Figure 3B).

[0031] A fusion protein of Mkl1 or Mkl2 and eGFP was incorporated into a Tet-On induction expression system, then incorporated into an adenovirus vector to produce a Tet-On adenovirus, which was expressed in myotubes. Myotubes were cultured in DMEM (Nacalai Tesque) containing 10% FBS (Gibco) on a dish coated with Geltrex® LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Thermo Fisher Scientific). After the C2C12 cells reached confluence, the DMEM (Nacalai Tesque) was replaced with DMEM (Nacalai Tesque) containing 2% Horse Serum (Gibco) to initiate differentiation. On day 3 of differentiation induction, adenovirus was added to the culture medium for infection, and doxycycline (wako) was added the following day to express Mkl1-eGFP and Mkl2-eGFP (Figure 3C). It was revealed that in myotubes, Mkl1 is localized in the nucleus, while Mkl2 is localized in the cytoplasm.

[0032] Using Tet-On adenovirus, myotubes expressing Mkl2-eGFP were subjected to electrical pulse stimulation (EPS) using an electrical stimulator, inducing periodic contractions in the myotube cells (Figure 3D). Upon electrical stimulation (EPS in the figure), Mkl2 migrates from the cytoplasm to the nucleus. It is thought that the contraction of myotubes induced by electrical stimulation, i.e., muscle contraction associated with exercise, causes Mkl2 to migrate to the nucleus, increasing muscle mass and suppressing muscle atrophy.

[0033] Muscle mass changes under various conditions. Aging, along with exercise, is a factor involved in the increase or decrease of muscle mass. First, we analyzed the expression of exercise-induced muscle atrophy-causing genes, as well as Mkl1 and Mkl2, using RT-qPCR. We analyzed the expression of Atrogin-1, MuRF1, Mkl1, and Mkl2 in the gastrocnemius muscle of young (8 weeks old) and aged (119 weeks old) C57BL / 6J mice. Atrogin-1 and MuRF1 are genes already known to be associated with muscle atrophy (muscle atrophy-causing genes), and are ubiquitin ligases specific to skeletal muscle and cardiac muscle. In aged (119 weeks old) mice, increased expression of Atrogin-1 and MuRF1 was observed compared to young (8 weeks old) mice. On the other hand, a significant decrease in Mkl1 expression was observed (Figure 4A). In other words, it became clear that Mkl1 expression decreases with age and is negatively correlated with the expression of muscle atrophy-causing genes. The decrease in expression levels due to aging was more pronounced in Mkl1 compared to Mkl2.

[0034] The correlation between the expression levels of Mkl1 and Atrogin1, MuRF1, Foxo1, and Foxo3 in the gastrocnemius muscle of mice of various ages was analyzed (Figure 4B). The x-axis shows the expression level of Mkl1, and the y-axis shows the expression levels of Atrogin1, MuRF1, Foxo1, or Foxo3, respectively. RNA-seq data (GEO accession no: GSE145480) registered in the public database Gene Expression Omnibus was re-analyzed. For the re-analysis, each expression level was normalized by DESeq. A strong inverse correlation was observed between Mkl1 and the muscle atrophy-causing genes (Atrogin1, MuRF1). In addition, an even stronger inverse correlation was observed between the expression levels of Mkl1 and Foxo1 and Foxo3, suggesting that Mkl1 may also suppress the transcriptional activity of the glucocorticoid receptor (GR), a nuclear receptor that regulates the expression of Foxo1 and Foxo3.

[0035] Next, using a muscle atrophy model induced by sciatic nerve resection, we performed expression analysis of Atrogin-1, MuRF1, Mkl1, and Mkl2 by RT-qPCR. The sciatic nerve of the right hindlimb of 10-week-old C57BL / 6J mice was resected. Day 0 was defined as the day before resection, and samples were taken of the left and right tibialis anterior muscles at days 3 and 7 post-resection. Changes in the expression of the gene group responsible for muscle atrophy in the tibialis anterior muscle, Mkl1 and Mkl2, were analyzed. At day 0, no difference in gene expression was observed between the left and right sides. However, at days 3 and 7 post-resection, increased expression of Atrogin-1 and MuRF1 and decreased expression of Mkl1 were observed in the right tibialis anterior muscle (the side on which the resection occurred). Conversely, increased expression of Mkl2 was observed (Figure 5).

[0036] Furthermore, since muscle atrophy can also occur in cases of poor nutritional status, we analyzed the changes in expression of muscle atrophy-causing genes and their regulatory factors due to fasting. Nine-week-old C57BL / 6J male mice were fasted for 24 hours, euthanized after fasting, and their gastrocnemius muscles were sampled. Changes in the expression of muscle atrophy-causing genes, Foxo1 / 3 / 4 and Mkl1 / 2, in the gastrocnemius muscle were analyzed in the same manner. In fasted mice (Fast), the expression of Atrogin-1 and MuRF1, which are muscle atrophy-causing genes, was significantly increased compared to mice fed normally (Fed). In addition, Foxo1 and Foxo3, which regulate muscle atrophy-causing genes, also showed a significant increase in the fasting group, and an increasing trend was also observed in Foxo4. On the other hand, Mkl1 and Mkl2 expression was suppressed in the fasting group, with particularly pronounced suppression of Mkl1 expression (Figure 6).

[0037] It has long been known that steroid treatment, such as dexamethasone, can cause muscle atrophy. We analyzed whether the expression of steroid-induced muscle atrophy-causing genes, including the FoxO gene, changes occur when Mkl1 or Mkl2 is expressed using a Tet-On adenovirus vector during the induction of muscle atrophy-causing genes. Specifically, C2C12 cells were differentiated, infected with Tet-ON adenovirus incorporating Mkl1 or Mkl2 on day 3 of differentiation, and gene expression was induced by adding doxycycline on day 4 of differentiation, along with the addition of 10 μM hydrocortisone or 10 μM dexamethasone (Figure 7A). The results showed that the expression of Atrogin-1, MuRF1, and FoxO3 genes was enhanced by hydrocortisone and dexamethasone, and suppressed by Mkl1 or Mkl2 expression.

[0038] It is known that the expression of the Foxo gene is induced by the glucocorticoid receptor (GR) during muscle atrophy. We analyzed whether the suppression of Foxo gene expression by Mkl1 during muscle atrophy was due to suppression of the glucocorticoid receptor using a luciferase assay (Figure 7B). A luciferase reporter was constructed by incorporating a sequence (SEQ ID NO: 1) containing four glucocorticoid receptor response elements (GR-RE) into the pGL4.23 plasmid (Promega), and used for the analysis. Luciferase activity is induced by glucocorticoid receptor (GR) expression, but suppressed by Mkl1 and Mkl2 expression. Furthermore, even when glucocorticoid receptor (GR) activation was induced in the presence of dexamethasone, the suppressive effect of Mkl1 and Mkl2 was observed. Therefore, it is thought that Mkl1 and Mkl2 suppress muscle atrophy induced by fasting and steroids, partly through the suppression of glucocorticoid receptor transcriptional activity. These results suggest that Mkl1 and Mkl2 also regulate glucocorticoid receptors.

[0039] Furthermore, as shown in the in vivo experimental data below, Mkl1 expression induces cardiac hypertrophy, suggesting that it can induce both cardiomyocyte hypertrophy and cell division promotion through the regulation of glucocorticoid receptors. Therefore, it is believed that inducing Mk11 / 2 expression can be applied to regenerative medicine, such as myocardial regeneration in myocardial infarction.

[0040] As shown above, in muscle atrophy resulting from various factors such as aging, disuse, neurogenic diseases, fasting, and steroid administration, Mkl1 expression shows an inverse correlation with the expression of muscle atrophy-causing genes. Furthermore, it was revealed that Mkl1 and Mkl2 regulate muscle atrophy-causing genes through the suppression of glucocorticoid receptors, Foxo1 / 3 / 4. In addition, since an increase in Mkl1 / 2 is observed with exercise, it is thought that Mkl1 / 2 controls muscle strengthening and suppression of muscle atrophy (see Figure 13).

[0041] Mkl1 and Mkl2 regulate muscle atrophy-causing genes via Foxo1 / 3 / 4 and glucocorticoid receptors (GR), but their behavior and effects on aging, sciatic nerve transection, fasting, and steroid administration differ. Furthermore, their intracellular localization also differs; Mkl2 resides in the cytoplasm and translocates to the nucleus upon exercise, while Mkl1 is localized in the nucleus. These results indicate that while Mkl1 and Mkl2 play similar roles in regulating muscle atrophy-causing genes, they are regulated differently and act on muscle atrophy caused by different factors. Further analysis is needed, but pharmaceutical compositions that increase Mkl2, which translocates to the nucleus upon exercise and exerts its function, are likely to be suitable as pharmaceutical compositions or supplements administered from the pre-disease stage to enhance the effects of rehabilitation or prevent sarcopenia. Furthermore, since Mkl1 is localized in the cell nucleus, it is thought to act more directly on muscle atrophy, and therefore may be effective against more severe muscle atrophy. For this reason, it is preferable to develop therapeutic drugs for muscle atrophy, or preventive drugs and supplements for pre-disease conditions, taking into account the characteristics of Mkl1 and Mkl2.

[0042] Since Mkl1 / 2 are thought to regulate muscle enhancement and atrophy through the suppression of FoxO, we analyzed the regulatory mechanism. We created a luciferase reporter construct (SEQ ID NO: 2) containing a FoxO response sequence consisting of a repeating sequence of the Foxo1 binding motif TGTTTAC, and analyzed the effects of Mkl1 and Mkl2 on the transcriptional activity of Foxo1 / 3 / 4. The luciferase reporter construct and each gene were introduced into 293FT cells (Thermo Fisher Scientific) by transfection using Polyethylenimine Max (MW 40,000) (Polysciences). Luciferase activity was measured by harvesting cells 48 hours after transfection, adding a luminescent substrate to the cell extract, and measuring the luminescence value using a GloMax® Discover Microplate Reader. Regardless of whether the Foxo1 / 3 / 4 gene was expressed, expressing either Mkl1 (Figure 8A, upper panel) or Mkl2 (Figure 8A, lower panel) resulted in a significant decrease in transcriptional activity.

[0043] Furthermore, since Foxo proteins are known to be inactivated by the PI3K / Akt pathway, Foxo1, Foxo3, and Foxo4 were activated in the presence of LY294002, an inhibitor of the PI3K / Akt pathway, and their activity was analyzed by luciferase assay (Figure 8B). LY294002 enhanced the transcriptional activity of Foxo1, Foxo3, and Foxo4, but this activation was suppressed by the expression of Mkl1 or Mkl2.

[0044] Furthermore, C2C12 cell lines were differentiated into myotubes, and Foxo1 or Foxo3 and Mkl1 or Mkl2 were introduced via adenovirus vectors and overexpressed. mRNA expression of muscle atrophy-causing genes was analyzed by RT-qPCR (Figure 8C, 8D left). Protein expression was also analyzed by Western blotting (Figure 8C, 8D right). Four days after induction of myotube differentiation, 20 μM of the PI3K inhibitor LY294002 (Fujifilm Wako Pure Chemical Corporation) was added to the culture medium and treated for 24 hours before analysis. Akt inhibits nuclear translocation and suppresses its activity by phosphorylating Foxo1 / 3 / 4. By adding the PI3K inhibitor LY294002, phosphorylation of Foxo1 / 3 / 4 is inhibited, leading to accumulation in the nucleus and maximal enhancement of its activity.

[0045] Whether Foxo1 was introduced and Mkl1 or Mkl2 was expressed (Figure 8C), or Foxo3 was introduced and Mkl1 or Mkl2 was expressed (Figure 8D), both Mkl1 and Mkl2 suppressed the expression of Atrogin-1 or MuRF1, which are muscle atrophy-causing genes. Furthermore, the suppressive effect of Mkl1 and Mkl2 was observed even in the presence of the PI3K inhibitor LY294002, indicating that FoxO suppresses gene expression regardless of whether the PI3K / Akt pathway is inhibited or not.

[0046] C2C12 cells were differentiated, and myotubes were treated with 20 μM PI3K inhibitor LY294002 and 10 μM Mkl1 / 2 nuclear localization inhibitor CCG-1423 for 24 hours on day 4 of induction. The expression of muscle atrophy-causing genes was analyzed by RT-qPCR (Figure 8E left) or Western blotting (Figure 8E right). As described above, the addition of LY294002 activated the FoxO gene group, and the addition of CCG-1423 inhibited the nuclear localization of Mkl1 and Mkl2. Therefore, the activity of the FoxO gene group was maximized. The expression of Atrogin-1 or MuRF1, which are muscle atrophy-causing genes, was enhanced by treatment with LY294002 and CCG-1423. Furthermore, an additive effect was observed when both were added simultaneously.

[0047] Next, we suppressed the expression of Mkl1 and Mkl2 using artificial microRNAs (miRNAs) and analyzed the expression of gene groups causing muscle atrophy (Figure 8F). The miRNAs used were constructed by linking three hairpin-structured miRNAs in tandem and regulating their expression via Tet-ON (Figure 8F, upper left). We used miRNAs for Mkl1 (Mkl1#1: SEQ ID NO: 3, Mkl1#2: SEQ ID NO: 4) and Mkl2 (Mkl2#1: SEQ ID NO: 5, Mkl2#2: SEQ ID NO: 6). By expressing the respective miRNAs for Mkl1 and Mkl2, the expression of Mkl1 or Mkl2 was specifically suppressed (Figure 8F, upper right). Furthermore, RT-qPCR (Figure 8F, lower left) and Western blotting (Figure 8F, lower right) showed that suppressing the expression of either Mkl1 or Mkl2 using miRNA resulted in increased expression of Atrogin-1 or MuRF1, which are genes responsible for muscle atrophy. These analyses revealed that Mkl1 and Mkl2 expression suppresses FoxO transcriptional activity, thereby suppressing the expression of genes responsible for muscle atrophy.

[0048] Furthermore, C2C12 cells were differentiated to form myotubes, and the effects of introducing a fusion protein of Foxo1 and eGFP (Foxo1-eGFP), Mkl1, or Mkl2 via adenovirus were analyzed (Figure 8G, top panel). While the introduction of Foxo1-eGFP induced myotube atrophy, simultaneous expression of Mkl1 or Mkl2 maintained myotubes and suppressed muscle atrophy. Additionally, Foxo3 and GFP were introduced separately via adenovirus, and the effects of Mkl1 or Mkl2 were analyzed (Figure 8G, bottom panel). Mkl1 and Mkl2 were similarly found to have an inhibitory effect on muscle atrophy when Foxo3 was expressed. Note that eGFP was used for the purpose of visualizing myotubes.

[0049] Next, we investigated whether the suppression of Foxo1 by Mkl1 and Mkl2 was due to direct binding, using immunoprecipitation (Figure 9A) and split luciferase assays (Figures 9B, 9C). We prepared Foxo1 with a Flag tag attached to the N-terminus and Mkl1 with an HA tag attached to the C-terminus (Figure 9A, upper left), Mkl1 with a Flag tag attached to the N-terminus and Foxo1 with an HA tag attached to the N-terminus (Figure 9A, upper right), Foxo1 with a Flag tag attached to the N-terminus and Mkl2 with an HA tag attached to the N-terminus (Figure 9A, lower left), and Mkl2 with a Flag tag attached to the N-terminus and Foxo1 with an HA tag attached to the N-terminus. We mixed each combination of the two and transfected 293FT cells with Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher Scientific) to induce expression. Two days later, the cells were collected and immunoprecipitation was performed with anti-FLAG antibody to analyze whether co-precipitation occurred. Since co-precipitation occurred with anti-FLAG antibody in all combinations, it was shown that Foxo1, Mkl1, and Mkl2 are directly bound to each other.

[0050] Next, the protein interaction (PPI) between Foxo1 and Mkl1 or Mkl2 was analyzed using a split luciferase assay (NanoBiT® PPI Starter Systems, Promega) (Figure 9B). Plasmids with N-terminal LgBiT tag, C-terminal LgBiT tag, N-terminal SmBiT tag, and C-terminal SmBiT tag were prepared for Foxo1, Mkl1, and Mkl2, respectively, and the interactions between each protein were analyzed. In Figure 9B, the SmBiT tag is denoted as Sm. 293FT cells were seeded in a 96-well white microplate, and the following day, plasmids with LgBiT tags and plasmids with SmBiT tags were cotransfected. For background signal measurement, HaloTag plasmids (Sm-HT) with SmBiT tags were used instead of SmBiT-tagged Foxo1, Mkl1, and Mkl2. 24 hours after transfection, a luminescent substrate was added, and the luminescence value was measured. Regardless of whether the construct used had the NanoBiT tag attached to the N-terminus or the C-terminus, a significant signal could be detected using a combination of Foxo1 and Mkl1 or Mkl2.

[0051] Furthermore, we analyzed the protein interactions between Foxo3 or Foxo4 and Mkl1 or Mkl2 (Figure 9C). Here, we show an example of analyzing protein-protein interactions by adding LgBiT tags to Mkl1 and Mkl2, and SmBiT tags to Foxo3 and Foxo4. We were able to detect signal values ​​above the background signal level with the combination of Mkl1 or Mkl2 and Foxo3 or Foxo4. The detection of a significant signal with the combination of Foxo1 / 3 / 4 and Mkl1 or Mkl2 indicates that Foxo1 / 3 / 4 directly binds to Mkl1 or Mkl2. In other words, the direct binding of Mkl1 and Mkl2 to Foxo1 / 3 / 4 was also shown by the split luciferase assay.

[0052] We analyzed whether changes occurred in the binding of Foxo1 to Mkl1 and Mkl2 using a system that inhibits the translocation of Mkl1 and Mkl2 to the nucleus in the presence of CCG-1423 (Figure 9D). We analyzed combinations in which Foxo1 was tagged with an LgBiT tag and Mkl1 (upper panel of Figure 9D) or Mkl2 (lower panel of Figure 9D) was tagged with an SmBiT tag. When nuclear translocation of Mkl1 and Mkl2 was inhibited by CCG-1423, a decrease in signal values ​​was observed, indicating a significant decrease in the binding of Foxo1 to Mkl1 and Mkl2. Therefore, it is suggested that Mkl1 and Mkl2 directly bind to Foxo1 in the nucleus and suppress its transcriptional activity.

[0053] To determine which regions of Foxo1 and Mkl2 are necessary for binding, we created N-terminal deletion mutants and analyzed them using a split luciferase assay. We created and analyzed deletion mutants of Foxo1 (Figure 10A, top panel) and Mkl2 (Figure 10A, bottom panel). Foxo1 lacking the Forkhead domain showed significantly reduced binding to Mkl2 (Figure 10A, top panel). The Forkhead domain is the DNA-binding domain of Foxo1 and is necessary for Foxo1 to directly bind to the cis-regulatory element present on gene promoters and enhancers. The result that the Forkhead domain is necessary for binding between Mkl2 and Foxo1 suggests that Mkl2 inhibits Foxo1's ability to bind to DNA by binding to its DNA-binding domain, thereby suppressing Foxo1's transcriptional activity.

[0054] On the other hand, in a split luciferase assay using a series of deletion mutants of Mkl2, the reduction in signal was more gradual even with only the Mkl2 transcriptional activation domain (TAD) compared to the full-length Mkl2, indicating that the TAD domain binds to Foxo1 (Figure 10A, bottom). Furthermore, the Mkl2 TAD domain alone was able to significantly suppress the transcriptional activity of Foxo1 (Figure 10B, left). In addition, since the TAD domain also showed an inhibitory effect on the fusion protein of the Foxo1 Forkhead domain and VP16 (Figure 10B, right), it is thought that the TAD domains of Mkl1 and Mkl2 bind to the DNA-binding domain of Foxo1 and suppress the transcriptional activity of Foxo1. Normally, the TAD domain is responsible for transcriptional activation, but a new mode of suppression has been revealed in which it inhibits and suppresses Foxo1's DNA binding by binding to the DNA-binding domain of Foxo1.

[0055] C2C12 cells were differentiated into myotubes, and the binding of Foxo3 to the genomic region surrounding muscle atrophy-causing genes in the presence of Mkl1 or Mkl2 was analyzed by chromatin immunoprecipitation (ChIP) assay. Adenovirus-tagged Foxo3 (Foxo3-HA), Mkl1, or Mkl2 was expressed in myotubes differentiated from C2C12 cells. 20 μM of the PI3K inhibitor LY294002 was added to induce nuclear translocation of Foxo3. Subsequently, the myotubes were fixed with 1% formalin, chromatin was extracted using Lysis buffer, and the chromatin was sheared by sonication. HA-tagged antibody (Proteintech) or control antibody was conjugated to Dynabeads® Protein A (Thermo Fisher Scientific), and chromatin immunoprecipitation (ChIP) was performed using this. After washing with RIPA buffer, the samples were heated to 65°C to remove crosslinking. After RNase A treatment and Proteinase K treatment, the genomic DNA was purified by phenol-chloroform extraction and ethanol precipitation. The enrichment rate of the purified genomic DNA was analyzed by qPCR using a primer set that detects Foxo3 binding sites around muscle atrophy-causing genes. The binding sites of Foxo3 around the Atrogin-1 and MuRF1 genes were quantified by qPCR (Figure 10C). It was observed that the binding of Foxo3 to Atrogin-1 or MuRF was significantly reduced when Mkl1 or Mkl2 was exogenously expressed. In other words, the ChIP assay also showed that the action of Foxo3 is inhibited by the direct binding of Mkl1 or Mkl2 proteins to the Foxo protein.

[0056] These results suggest that increased expression of the Mkl1 / 2 protein inhibits the function of the FoxO protein as a transcription factor by binding to it, thereby inhibiting the induction of expression of muscle atrophy-causing genes. As a result, it was shown to have an inhibitory effect on muscle atrophy. In other words, if the expression of Mkl1 / 2 can be enhanced, muscle atrophy can be suppressed or muscle can be strengthened. Since Mkl1 / 2 is located upstream of FoxO, it is thought that controlling it as a molecular target can suppress muscle atrophy more sensitively.

[0057] We analyzed whether enhancing Mkl1 / 2 expression could suppress muscle atrophy or promote muscle growth using an in vivo system with mice. Muscle-specific adeno-associated virus was prepared using the AAV-MAX Helper-Free AAV Production System Kit (Thermo Fisher Scientific). VPC2.0 cells were transfected with Helper plasmid, Rep / Cap plasmid, and Transfer plasmid, and after 72 hours, AAV was extracted using citrate buffer (55 mM citrate, 55 mM sodium citrate, 800 mM NaCl). After neutralization with HEPES (pH 8.0), PEG precipitation was performed. After resuspending, the solution was treated with benzonase and purified by density gradient centrifugation using Optiprep. The purified AAV was administered to mice by tail injection after replacing the buffer with PBS.

[0058] The transfer plasmid was created by removing all inter-ITR sequences from the pAAV-CMV Vector (TaKaRa) and incorporating the Mhck7 promoter, a hybrid promoter of the mouse muscle creatine kinase gene promoter and the mouse α-myosin heavy chain gene promoter, along with the SV40polyA transcription termination signal. Furthermore, eGFP, Akaluc, or Mkl1 sequences were incorporated. DNA templates for the Mhck7 promoter, Akaluc, and Mkl1 were created by artificial gene synthesis (Integrated DNA Technologies). Codon optimization was performed for Akaluc and Mkl1. The Rep / Cap plasmid encoding MyoAAV.2A was created by inverse PCR based on pUCmini-iCAP-PHP.S(Addgene) by substituting the sequence SAQQAVRTSL (SEQ ID NO: 7) in hypervariable region VIII with GPGRGDQTTL (SEQ ID NO: 8). The helper plasmid used was pHelper (TaKaRa). Furthermore, in the following experiments, when introducing genes such as eGFP and Akaluc using adeno-associated virus, plasmids were constructed with the same configuration, except that the respective gene sequences were used.

[0059] First, we investigated whether foreign genes could be introduced into mouse muscles using muscle-targeting adeno-associated viruses, specifically using Akaluc or eGFP. Mice were injected via tail intravenous injection at a rate of 1 × 10⁶. 11 MyoAAV.2A Mhck7-Akaluc was administered to mice, and Akaluc expression was measured over time using IVIS (Figure 11A). In virus-administered mice, expression was observed on day 4, and Akaluc expression increased over time, becoming systemic from day 7 onwards, and showing strong expression from day 14 onwards. Furthermore, it was observed that Akaluc expression was specifically and strongly induced in the muscle and heart.

[0060] Furthermore, after administering MyoAAV.2A Mhck7-eGFP, eGFP expression was analyzed 38 days after administration by extracting the gastrocnemius muscle, tibialis anterior muscle, plantaris muscle, extensor digitorum longus muscle, and heart. As a result, eGFP expression was observed in all muscles (Figure 11B). These results suggest that it is possible to induce expression in muscles by introducing Mkl1 or Mkl2 using this system.

[0061] We introduced the Mkl1 gene into mice using adeno-associated virus and analyzed whether the expression of Foxo1, Foxo3, and Foxo4 changed during fasting (Figure 11C). Exogenous expression of Mkl1 suppressed the induction of Foxo1 and Foxo3 gene expression induced by fasting. Therefore, it was shown that the expression of Foxo1 and Foxo3 is regulated by Mkl1. However, since Foxo4 expression did not increase during fasting, the effect of exogenously expressed Mkl1 could not be confirmed. In the figure, VEH means administration of the solvent (Vehicle) only.

[0062] Furthermore, we analyzed in vivo whether Mkl1 / 2 has an inhibitory effect on steroid-induced muscle atrophy (Figure 11D). Mkl1 was introduced into the muscles of 9-week-old mice using adeno-associated virus, and after 12 weeks, dexamethasone sodium phosphate was administered intraperitoneally at a concentration of 25 mg / kg. The following day, the gastrocnemius muscle was removed, and the expression of the muscle atrophy-causing genes, Foxo1, Foxo3, and Foxo4, was analyzed. In vivo, Mkl1 expression suppressed the expression of MuRF1, Foxo1, and Foxo3, and a tendency towards suppression was also observed in Atrogin-1 and Foxo4. This demonstrated that Mkl1 expression has an inhibitory effect on muscle atrophy in vivo caused by steroids such as dexamethasone.

[0063] We investigated whether strengthening Mk1 / 2 would suppress muscle atrophy and lead to increased muscle mass. 1×10 11 MyoAAV.2A Mhck7-Mkl1 was administered intravenously via tail injection, and the gastrocnemius muscle was removed and weighed 12 weeks later (Figure 11E). In individuals expressing Mkl1, muscle hypertrophy was observed visually, and a significant increase in muscle weight was also demonstrated.

[0064] Furthermore, we analyzed the effects on the heart (myocardium) (Figure 11F). 1 × 10 11 vg, 2×10 11 MyoAAV.2A Mhck7-Mkl1 (vg) was administered intravenously via tail injection, and the heart was removed 12 weeks later for weight analysis. 1 × 10 11 When the virus was administered via vg, no difference was observed compared to the control administered with PBS, but 2 × 10 11 When administered via VG, myocardial hypertrophy was observed visually, and a significant increase in cardiac weight was demonstrated. This myocardial hypertrophy is thought to be due to both the enlargement of the cardiomyocytes themselves and an increase in the number of cardiomyocytes through cell division.

[0065] The results above demonstrate that introducing and expressing Mkl1 in muscle using adeno-associated virus suppresses the expression of muscle atrophy-causing genes and further enlarges skeletal and cardiac muscle, indicating a therapeutic effect against muscle atrophy. Since decreased expression of Mkl1 and Mkl2 was observed in muscle atrophy caused by various factors such as aging, disuse, neurogenicity, malnutrition, and steroid administration, the introduction and expression of Mkl1 and Mkl2 using viral vectors is considered to have a therapeutic effect against muscle atrophy caused by these factors. In this study, Mkl1 / 2 was introduced using adeno-associated virus, but expression in muscle may also be performed using other viral vectors or expression systems. Furthermore, since even TAD, a part of the Mkl2 protein, can suppress Foxo1 expression, it is not necessary to express the full length of Mkl1 / 2; it is thought that expressing the region containing TAD in muscle is sufficient to achieve a good effect.

[0066] Next, we showed that Mkl1 / 2 proteins are rapidly ubiquitinated and degraded. We also analyzed whether the amount of Mkl1 / 2 proteins could be increased by suppressing proteasome degradation. In myotubes differentiated from C2C12 cells, Mkl1 and Mkl2 with HA tags added using the Tet-On system were expressed, and after doxycycline (Dox) removal, changes in intracellular protein levels were analyzed by Western blotting (Figure 12A). Both Mkl1 and Mkl2 had short half lives, and Mkl1 in particular was shown to be a more unstable protein.

[0067] Therefore, myotubes differentiated from C2C12 cells were treated with the ubiquitin-proteasome inhibitor MG-132, and the amounts of Mkl1 and Mkl2 were analyzed (Figure 12B). Since the amount of both Mkl1 and Mkl2 proteins was stabilized by the ubiquitin-proteasome inhibitor, it is thought that they are constitutively degraded by the ubiquitin-proteasome. Thus, ubiquitin-proteasome inhibitors may be effective as pharmaceutical compositions that stabilize the amounts of Mkl1 and Mkl2 and suppress muscle atrophy.

[0068] A screening method for substances that can increase Mkl1 / 2 protein levels can be, for example, by exposing cultured cells that express Mkl1 / 2, such as cells established from muscle, to candidate compounds and confirming enhanced Mkl1 / 2 expression. Highly sensitive screening can be performed by creating and introducing constructs that fuse a fluorescent protein such as GFP or a reporter protein such as luciferase to the Mkl1 / 2 gene. Alternatively, a reporter gene such as luciferase can be knocked in downstream of the Mkl1 / 2 gene using genome editing to enable the expression of a fusion protein, and then the cells can be exposed to candidate compounds and screened using the expression of the reporter gene as an indicator. After candidate compounds are selected in cells, further selection of compounds can be performed using cells that can be induced into myotubes or primary myotubes. Furthermore, since muscle atrophy is caused by steroids such as dexamethasone, studies can be conducted using animal models in which muscle atrophy has been induced by steroids. As mentioned above, ubiquitin-proteasome inhibitors have been shown to suppress the degradation of Mkl1 / 2. Therefore, drugs that specifically inhibit the degradation of Mkl1 and Mkl2 by ubiquitin-proteasomes are considered promising candidates for muscle-building therapies.

[0069] As described above, the inventors have discovered novel target molecules in muscle atrophy and muscle enhancement. In muscle atrophy, the activation of GR and Foxo1 / 3 / 4 induces the expression of muscle atrophy-causing genes. Furthermore, the nuclear receptor GR also induces the expression of Foxo1 and Foxo3 upon activation. We have revealed that Mkl1 and Mkl2 suppress muscle atrophy by inhibiting the transcriptional activity of GR and Foxo1, Foxo3, and Foxo4 (Figure 13). From these results, it can be concluded that increasing the expression of Mkl1 / 2 can suppress and treat muscle atrophy caused by various factors for which there have been no effective therapeutic drugs until now.

Claims

1. Targeting Mkl1 and / or Mkl2, A muscle atrophy inhibitor and / or muscle enhancer characterized by containing a substance that increases the amount of Mkl1 and / or Mkl2 as an active ingredient.

2. A muscle atrophy inhibitor and / or muscle enhancer according to claim 1, characterized by increasing the amount of Mkl1 and / or Mkl2 in the nucleus.

3. The muscle atrophy inhibitor and / or muscle strengthening agent according to claim 1, characterized in that the muscle atrophy inhibitor is used for the treatment of muscle atrophy caused by aging, disuse, neurogenic causes, malnutrition, or steroid administration.

4. The muscle atrophy inhibitor and / or muscle enhancer according to claim 1, characterized in that Mkl1 and / or Mkl2 are introduced by a viral vector.

5. By suppressing the decomposition of Mkl1 and / or Mkl2, The muscle atrophy inhibitor and / or muscle enhancer according to claim 1, characterized by increasing the amount of Mkl1 and / or Mkl2.

6. A muscle atrophy inhibitor and / or muscle enhancer according to any one of claims 1 to 5, characterized in that the muscle is skeletal muscle or cardiac muscle.

7. The candidate compound was exposed to cultured cells, A screening method for muscle atrophy inhibitors and / or muscle enhancers, characterized by using an increase in the amount of Mkl1 and / or Mkl2 in cultured cells as an indicator.

8. A reporter protein is fused to the Mkl1 and / or Mkl2 proteins. A screening method for muscle atrophy inhibitors and / or muscle enhancers according to claim 7, characterized in that an increase in reporter protein is used as an indicator.

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