KR20100115910A - Methods for screening a therapeutic agent of heart failure, fibrosis and inflammation and compositions comprising the same - Google Patents

Abstract

PURPOSE: A composition containing Eya2 gene is provided to effectively prevent or treat heart failure, fibrosis or inflammation. CONSTITUTION: A method for screening a therapeutic agent for preventing or treating heart failure, fibrosis or inflammation comprises: a step of treating test material to cells having Eya2 gene; and a step of analyzing the expression of Eya2 gene. In case of increase of Eya2 gene expression, the test material is considered as a therapeutic agent for preventing or treating heart failure, fibrosis or inflammation. A composition for preventing or treating heart failure, fibrosis or inflammation contains Eya2 gene as an active ingredient.

Description

Methods for Screening a Therapeutic Agent of Heart Failure, Fibrosis and Inflammation and Compositions Comprising the Same}

The present invention relates to a method for screening a therapeutic agent for heart failure, fibrosis or inflammation and a composition comprising the same.

Mechanical stress exerted on the heart wall by pressure overload, which can be caused by various pathological injuries, including aortic stenosis, hypertension and myocardial infarction, causes cardiac hypertrophy. do. This process, marked by the enlargement of cardiomyocytes, the accumulation of contractile proteins, and the reorganization of myofibrillar structures, is intended to relieve stress on the myocardial wall by increasing cardiac output [1]. . Although taking into account the benefits at an early stage, cardiac hypertrophy often progresses to heart failure, increasing the risk of sudden death. Cardiomegaly is thus an independent and major risk factor for cardiovascular morbidity and mortality [2]. Intracellular and molecular processes associated with cardiac hypertrophy have been the target of a very wide range of studies. Various studies report the integration of multiple pro- and anti-hypertrophic signaling pathways to regulate cardiac hypertrophy processes [3-5]. Although specific knowledge of pro-hypertrophic signaling pathways is accumulating, only fragmentary facts are known in the anti-hypertrophic signaling pathway. Based on the hypothesis that unique signal pathways are activated during the course of cardiac hypertrophy-regression, we previously isolated genes associated with anti-hypertrophic signal pathways using a cardiac hypertrophy-regressive surgical model [6]. The inventors found that one of the isolated transcription factors, eyes absent 2 (Eya2), inhibits phenylephrine (PE) -induced myocardial hypertrophy, suggesting that Eya2 is a novel anti-hypertrophic signal pathway. It is a component [6].

The eye absent (eya) gene was first identified in a study of eye development in fruit flies. Loss of function of the eya gene typically results in damage to the entire eye [7, 8], and directed expression of the gene leads to the formation of ectopic eyes [9-11]. Mammals have four Eya homologs, Eya1-4 [12, 13]. Eya proteins, as transcription factors, have a variety of amino-terminal domains with transcriptional activation and highly conserved carboxyl-terminal domains that mediate interaction with other transcription factors such as Six and Dach [14-16]. Interactions between Eya and Six or Eya and Dach, which are susceptible to forming multi-subunit transcriptional complexes, are important not only for fruit fly eye development, but also for mammalian organogenesis. Recently, extensive research has been conducted on the function of individual Eya genes in mammals. Mutations in Eya1 and Eya4 cause branch- o -renal syndrome, an autosomal dominant disease characterized by branching arch abnormalities, hearing loss and kidney defects. [17] Furthermore, mutations in Eya4 result in autosomal dominant syndrome characterized by dilated cardiomyopathy and heart failure preceded by hearing loss in the perceptual organs [18]. Although the association of the Eya2 and Eya3 genes with human diseases is not well established, the expression of the Eya2 gene is increased in ovarian cancer and promotes tumor growth [19].

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The inventors have sought to find new targets of therapeutic agents for preventing or treating heart failure, fibrosis or inflammation. As a result, the inventors found that overexpressing the Eya2 (eyes absent 2) gene inhibits harmful cardiac remodeling (eg, heart failure), including fibrosis and inflammation, and activates the PI3K / Akt / mTOR signaling pathway. By developing a screening method, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a method for screening a prophylactic or therapeutic agent for heart failure, fibrosis or inflammation.

Another object of the present invention to provide a composition for preventing or treating heart failure, fibrosis or inflammation.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for screening a prophylactic or therapeutic agent for heart failure, fibrosis or inflammation, comprising the following steps: (a) a cell comprising an Eya2 (eyes absent 2) gene Treatment of the test substance in the laboratory; And (b) analyzing the expression of the Eya2 gene, when the test substance increases the expression of the Eya2 gene is determined to prevent or treat heart failure, fibrosis or inflammation.

The inventors have sought to find new targets of therapeutic agents for preventing or treating heart failure, fibrosis or inflammation. As a result, the inventors found that overexpressing the Eya2 (eyes absent 2) gene inhibits harmful cardiac remodeling (eg, heart failure), including fibrosis and inflammation, and activates the PI3K / Akt / mTOR signaling pathway. One screening method was developed.

The present invention provides a method for screening a substance for preventing or treating heart failure, fibrosis or inflammation.

According to the method of the present invention, first, a sample to be analyzed is contacted with a cell containing the Eya2 gene or protein. Preferably, the cell comprising the Eya2 gene or protein is a cell derived from the heart. As used to refer to the screening method of the present invention, the term "test material" refers to an unknown material used in screening to examine whether the amount of Eya2 gene expression or Eya2 protein is affected. The test substance includes, but is not limited to, chemicals, nucleotides, antisense-RNAs, small interference RNAs (siRNAs), and natural extracts.

Subsequently, the expression level of the Eya2 gene or the amount of the Eya2 protein is measured in the cells treated with the test substance. As a result of the measurement, if the expression level of the Eya2 gene or the amount of the Eya2 protein is measured to be up-regulated, the test substance may be determined as a prophylactic or therapeutic agent for heart failure, fibrosis or inflammation.

Measurement of the expression level change of the Eya2 gene can be carried out through various methods known in the art. For example, RT-PCR (Sambrook et al., Molecular Cloning. A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)), Northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine , 102-108, CRC press), hybridization reaction using cDNA microarray (Sambrook et al., Molecular Cloning.A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)) or in situ hybridization reaction (Sambrook et al. , Molecular Cloning.A Laboratory Manual , 3rd ed.Cold Spring Harbor Press (2001)).

In case of carrying out the RT-PCR protocol, first, total RNA is isolated from cells treated with the test substance, and then the first-chain cDNA is prepared using oligo dT primer and reverse transcriptase. Subsequently, the first chain cDNA is used as a template, and PCR reaction is performed using an Eya2 gene-specific primer set. Eya2 gene-specific primer sets are illustrated in the examples below. Then, PCR amplification products are electrophoresed, and the formed bands are analyzed to measure changes in the expression level of the Eya2 gene.

According to the screening method of the present invention, the Eya2-encoding nucleotide sequence is preferably present in a suitable expression construct. In the expression construct, the Eya2 gene is preferably operably linked to a promoter. As used herein, the term “operably linked” refers to a functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and other nucleic acid sequences, whereby such regulation The sequence will control the transcription and / or translation of said other nucleic acid sequence. In the present invention, the promoter bound to the Eya2 gene is preferably capable of controlling transcription of the Eya2 gene by operating in animal cells, more preferably mammalian cells, and promoters and mammalian cells derived from mammalian viruses. Promoters derived from the genome of, for example, the CMV (cytomegalo virus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, RSV promoter, EF1 alpha promoter, metallothionine Promoter, beta-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene, promoter of human GM-CSF gene and alpha-myosin-heavy chain promoters of the (α-MHC) gene, including but not limited to. Most preferably, α-MHC promoter.

Preferably, the expression construct used in the present invention comprises a poly anenylation sequence. More preferably, the polyaninylation sequence is a plastic field hormone terminator (Gimmi, ER, et al., Nucleic Acids Res. 17: 6983-6998 (1989)), an SV40-derived polyadenylation sequence (Schek, N, et al. , Mol. Cell Biol. 12: 5386-5393 (1992)), HIV-1 polyA (Klasens, BIF, et al., Nucleic Acids Res. 26: 1870-1876 (1998)), β-globin polyA (Gil , A., et al, Cell 49: 399-406 (1987)), HSV TK polyA (Cole, CN and TP Stacy, Mol. Cell. Biol. 5: 2104-2113 (1985)) or polyomavirus polyA ( Batt, D. B and GG Carmichael, Mol. Cell. Biol. 15: 4783-4790 (1995).

Changes in the amount of Eya2 protein can be carried out through various immunoassay methods known in the art. For example, changes in the amount of Eya2 protein include, but are not limited to, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assay, and sandwich assay. It is not. The immunoassay or method of immunostaining is described in Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, when the method of the present invention is carried out in accordance with radioimmunoassay methods, antibodies labeled with radioisotopes (eg, C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ) may be used to detect Eya2 protein. Can be.

When the method of the present invention is carried out in an ELISA manner, certain embodiments of the present invention comprise the steps of: (i) coating an unknown cell sample (eg, heart) lysate to be analyzed on the surface of a solid substrate; (Ii) reacting said cell lysate with an antibody against Eya2 protein as a primary antibody; And (iii) reacting the resultant of step (ii) with the secondary antibody to which the enzyme is bound.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most preferably microtiter plates.

Enzymes bound to the secondary antibody include, but are not limited to, enzymes catalyzing color reaction, fluorescence, luminescence or infrared reaction, for example, alkaline phosphatase, β-galactosidase, hose Radish peroxidase, luciferase and cytochrome P ₄₅₀ . When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as a substrate Chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis) if colorimetric substrates such as -B1-phosphate) and enhanced chemifluorescence (ECF) are used, and horse radish peroxidase is used -N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amflex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o -phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS substrates such as (phenzaine methosulfate) may be used. All.

When the method of the invention is carried out in a capture-ELISA mode, certain embodiments of the invention comprise the steps of: (i) coating the surface of a solid substrate with an antibody against the Eya2 protein as a capturing antibody; (Ii) reacting the capture antibody with a cell sample (eg, FLS); (Iii) reacting the result of step (ii) with a detecting antibody having a label that generates a signal and which specifically reacts with the Eya2 protein; And (iv) measuring a signal originating from said label.

The detection antibody carries a label which generates a detectable signal. The label may include chemicals (eg biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P ₄₅₀ ), radioactive substances (eg C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ), fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent, and fluorescence resonance energy transfer (FRET). Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999.

Measurement of the final enzyme activity or signal in the ELISA method and the capture-ELISA method can be carried out according to various methods known in the art. Detection of these signals allows for qualitative or quantitative analysis of the Eya2 protein. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used.

According to a preferred embodiment of the invention, the fibrosis or inflammation is fibrosis or inflammation in the heart and causes heart failure.

According to a preferred embodiment of the invention, the analysis of gene expression in step (b) of the present invention is carried out by measuring the RNA or Eya2 protein of the Eya2 gene (see Figure 6).

According to a preferred embodiment of the invention, the method further comprises analyzing the expression of genes involved in the PI3K / Akt / mTOR signaling pathway. More preferably, expression analysis of genes involved in the PI3K / Akt / mTOR signaling pathway is performed by detecting the expression of the gene (eg, PI3K, pAKT or pp70S6K protein) via western blotting. Such expression analysis can be carried out very simply and easily by those skilled in the art. Based on this, the inventors confirmed that Eya2 gene responds to changes in ventricular wall stress and overexpression of Eya2 inhibits the development of pressure overload-induced cardiac hypertrophy and heart failure. This means that Eya2 activates a novel anti-hypertrophic signaling pathway, which in turn provides a novel strategy to treat cross-talk and deleterious cardiac remodeling with the PI3K / Akt / mTOR signal cascade.

According to another aspect of the present invention, the present invention provides a composition for preventing or treating heart failure, fibrosis or inflammation, including Eya2 (eyes absent 2) gene as an active ingredient.

In the present invention, the Eya2 gene of the present invention provides a novel therapeutic target for preventing or treating heart failure, fibrosis or inflammation.

As used herein, the term “heart failure” refers to a disease, condition or condition that is damaged so that the structure or function of the heart does not supply sufficient blood flow as needed by the body. The pathogenesis of heart failure includes myocardial infarction and other forms of hemorrhagic heart disease, hypertension, valve heart disease and cardiomyopathy (McMurray, JJ and Pfeffer, MA, "Heart failure", Lancet , 365 (9474): 187789 (2005)).

Heart failure is associated with fibrosis and inflammation. Cardiac fibrosis is a disease, condition or condition in which the thickness of the heart plate is abnormally thickened due to inadequate proliferation of cardiac fibroblasts. In normal cases, fibrocytes secrete collagen to structurally support the heart. This overactivation of the secretory process leads to enlargement of the valves, leading to fibrosis of the valves where white tissue builds up primarily on the tricuspid valve and pulmonary valve. It causes enlargement and loss of flexibility, leading to valve abnormalities and right-sided heart failure.

According to the present invention, Eya2 of the present invention inhibited the heart failure induced by pressure overload, blocked the progression of fibrosis and significantly reduced the level of the pro-inflammatory cytokine IL-1β (see FIG. 4). This indicates that Eya2 inhibits harmful remodeling of the heart, including fibrosis and inflammation. Therefore, the composition of the present invention comprising Eya2 as an active ingredient is very useful in preventing or treating heart failure, fibrosis or inflammation.

The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the present invention may be oral or parenteral (eg, intravenous, intraperitoneal, intramuscular, subcutaneous, or topical).

Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.001-100 mg / kg body weight per day.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or it may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for screening an agent for preventing or treating heart failure, fibrosis or inflammation.

(b) According to the present invention, Eya2 overexpression can inhibit the fibrosis and inflammation of the heart under prolonged pressure overload, as well as weakening of the wall, ventricular dilatation and deterioration of heart function.

(c) In the present invention, the composition of the present invention comprising the Eya2 gene as an active ingredient is effective in preventing or treating heart failure, fibrosis or inflammation.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Materials and Experimental Methods

Semi-quantitative RT-PCR

Total RNA was isolated using TRI reagent (Sigma). Reverse-transcription was performed by priming oligo-dT using ImProm II reverse-transcriptase (Promega). The following oligonucleotide primers were used for semi-quantitative RT-PCR: ANF, 5′-ACCTGCTAGACCACCTGGAGGAG-3 ′ and 5′-CTTTGGCTGTTATCTTCGGTACCG-3 ′; Eya1, 5′-ATGGAAATGCAGGATCTAACC-3 ′ and 5′-GGCTTGTTGCATTCCTGTGGT-3 ′; Eya2, 5′-ATGTTAGAAGTGGTGATCTCA-3 ′ and 5′-GCCTTGATAGAGCCCTGCCCT-3 ′; Eya3, 5′-ATGCAGGAACCAAGAGAACAG-3 ′ and 5′-GCTGTCCTGTCTGCGAGTAGG-3 ′; Eya4, 5′-ATGGAAGACACCCAGGACCTA-3 ′ and 5′-GCTGTCCTGTCTGCGAGTAGG-3 ′; GAPDH, 5′-TCCGTGTTCCTACCCCCAATG-3 ′ and 5′-GGGAGTTGCTGTTGAAGTCGC-3 ′. The template was subjected to 27 cycles of denaturation at 95 ° C. for 30 seconds, annealing at 58-60 ° C. for 30 seconds and extension at 72 ° C. for 30 seconds. PCR products were loaded on 1.2% agarose gel and electrophoresed at 100V for 20 minutes. PCR-amplified DNA products were quantified by comparison with standard curves calculated using known amounts of pcDNAEya1, pcDNAEya2, pcDNAEya3 and pcDNAEya4 as PCR templates. Each reference DNA was amplified using the PCR conditions described above. DNA gel image data were analyzed using QuantityOneTM software V6.1.1 (Bio-Rad, Hercules, CA).

Construction of Transgenic Mice

The full-length mouse Eya2 cDNA was constructed including 5.5 kb of human growth hormone 3′-UTR and alpha -myosin-heavy-chain ( α-MHC ) promoters and microinjected into FVB fertilized eggs. Southern blotting (Macrogen, Korea) was used to confirm the integration of the transplant gene. 8-10 week old transgenic mice and wild type controls were analyzed.

Transverse aorta constriction (TAC)

8-10 week old male mice (25-32 g) were used in the present invention. Mice were anesthetized by intraperitoneal injection of 0.5-0.7 ml of 1 × Avertin solution (combination of 2-2-2 tribromoethanol and tert-amyl alcohol). Mice were given air at a tidal volume of 0.1 ml and a respiratory rate of 120 times per minute (Harvard Apparatus). In order to observe the aortic arch, 2-3 mm sections with vertical cuts near the sternum were made. An overlayed 27-gauge needle was used to bind the transverse aortic arch between the innominate arteries and the left common carotid arteries and immediately remove the needle beyond the apparent site of constriction. At week 1, the mortality rate was less than 10%.

Histological Analysis of the Heart

Animals were sacrificed 4 or 12 weeks after TAC or Sham surgery. The heart was rested at the end-diastole and the left ventricle was separated from the right ventricle and weighed. Paraffin-embedded heart and glycol methacrylate-embedded (Technovit 8100, Germany) were cut into 4 μm and 1.5 μm slices, respectively, and stained with hematoxylin-eosin solution. In order to measure the surface area of islet myoblasts, appropriate cross-sections containing near-circular capillary profiles and nuclei were selected and observed with an Axiophot microscope (Carl Zeiss, Germany) and analyzed by AnalysisSiS3.2 software (Soft-Imaging). System, Germany). To determine the fibrosis site, the sectioned heart was stained with tricom staining. The fibrosis site was stained blue and normal tissue was stained red. The fibrosis site of the interstitial tissue was calculated by the ratio of the total fibrosis site of the interstitial tissue to the entire site of the segment. The sections were observed with an Axiophot microscope (Carl Zeiss, Germany) and analyzed using AnalysisSiS3.2 software (Soft-Imaging System, Germany).

Echocardiography

After anesthetizing the mouse, the chest area was depilated. Ultrasound cardiac examination was performed using a Powervision 6000 (TOSHIBA) device with a 12-MHz microprobe (PLM-1204AT, Toshiba). The heart was scanned using M-mode to search in a two-dimensional short-axis view. A frozen frame was printed using a video graphics printer (UP-895MD, Sony). Ventricular measurements were obtained using M mode.

Quantitative RT-PCR

Total RNA was isolated using TRI reagent (Sigma). Reverse-transcription was performed by priming oligo-dT using ImProm II reverse-transcriptase (Promega). PCR was performed using ABI PRISM Sequence Detector System 7500 (Applied Biosystems) using SYBR Green (TaKaRa) as fluorescence die and ROX (TaKaRa) as negative reference die. The following PCR primers were used: ANF, 5′-ACCTGCTAGACCACCTGGAGGAG-3 ′ and 5′-CTTTGGCTGTTATCTTCGGTACCG-3 ′; BNP, 5′-GCTGCTTTGGGCACAAGATAG-3 ′ and 5′-GGTCTTCCTACAACAACTTCA-3 ′; α-MHC, 5′-ACAATGCGGAAGTGGTGG-3 ′ and 5′-TCTTGCTACGGTCCCCTATG-3 ′; β-MHC, 5′-GACGAGGCAGAGCAGATCGC-3 ′ and 5′-GGGCTTCACAGGCATCCTTAGCC-3 ′; Collagen 1, 5′-CGAAGGCAACAGTCGCTTCA-3 ′ and 5′-GGTCTTGGTGGTTTTGTATTCGAT-3 ′; TGF-β2, 5′-AGCGCTACATCGATAGCAAG-3 ′ and 5′-TCCTGTCTTTGTGGTGAAGC-3 ′; IL-1β, 5′-TCCAGGATGAGGACATGAGCAC-3 ′ and 5′-GAACGTCACACACACCAGCAGGTT-3 ′; GAPDH, 5′-TCCGTGTTCCTACCCCCAATG-3 ′ and 5′-GGGAGTTGCTGTTGAAGTCGC-3 ′.

Western blot analysis

Heart tissue lysates were obtained by lysis with SDS-lysis buffer (1% SDS, 10 mM Tris-HCl, pH 7.4) and protease inhibitor cocktail (Boehringer Mannheim, Germany). 50 μg cardiac tissue lysates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Schleicher & Schuell, Germany). The membrane was blocked with 5% skim milk powder and reacted with antibodies against Eya2 (AbFrontier, Korea), Akt, phosoho-Akt, p70S6K (Cell Signaling, USA), PI3K (85α) and tubulin (Santa Cruz, USA). The reaction with the primary antibody was always carried out overnight in the cold room. Membranes were reacted with horseradish peroxidase (HRP) -bound secondary antibody (Jackson ImmunoResearch) and detected using a chemifluorescent substrate (PerkinElmer).

cDNA Microarrays and Genetic Analysis

For each experiment total RNA was extracted from five left ventricles and hybridized with Whole Mouse Genome Oligo Microarray (Agilent) containing 41,000 transcripts according to manufacturer's recommendations. The completed slides were scanned with an Agilent DNA microarray scanner and analyzed with Feature Extraction software (Agilent). Reproducibility of the obtained data was evaluated through scatter plot analysis of standardized probe signals. The remaining 4 data were analyzed in more detail by excluding the data with the lowest correlation coefficient among the 5 data at each point. The mean normalization method was used to further normalize the data. Samples were scaled to an average 2000 target density and then log converted by base two. Probes with less than 50% presence-call were removed [20]. Genes that were distinguishably expressed were selected based on the fold-change values and p-values obtained by Student's-test. Gazer ( http://integromics.kobic.re.kr/GAzer/index.faces ) [21] was used for gene analysis of gene expression data sets. Parametric Analysis of Gene Set Enrichment (PAGE) [22] was used to identify gene sets associated with specific phenotypes. Gene sets of BioCarta and KEGG pathways were obtained from the CGAP Pathway website (http: //cgap.nci.nih/gov/Pathways). Clustering and visualization of expression data were performed using cluster and Java treeview programs [23].

statistics

Data is expressed as mean ± SD. Comparison of mean values for each group was performed using one-way ANOVA (Statview V5.0, SAS), including Student's test or Bonferroni post-hoc test. P <0.05 was considered statistically significant.

Experiment result

Construction of Transgenic Mice Expressing Heart-Specific Eya2

We have previously found that Eya2 is induced in the regression of cardiac hypertrophy and that adenovirus-mediated overexpression of Eya2 in rat neonatal myoblasts inhibits PE-induced hypertrophy [6]. Mammals have four Eya homologues (Eya1-4). Since other Eya proteins could also inhibit PE-induced myoblast hypertrophy (no results), we could not determine whether Eya homologues were physiologically related to cardiac remodeling. We therefore monitored the expression of Eya homologues using semi-quantitative RT-PCR during the development and regression of cardiac hypertrophy. To ensure cardiac hypertrophy, transverse aortic stenosis (TAC) was applied for 14 days to obtain a heart (TAC14) or surgical removal of ligature after 14 days of stenosis to eliminate stenosis and after one day Was obtained (TAC-R1). Cardiac hypertrophy regressed markedly in TAC-R1 samples (no results shown). The expression of ANF was clearly increased in TAC14 samples and decreased in TAC-R1 samples, indicating that our RNA samples were valid (FIG. 1A). To determine the absolute amount of Eya transcript, we performed semi-sequential RT-PCR with TAC14 and TAC-R1 samples and an amount of Eya cDNA known as the concentration standard (FIG. 1A). Using Eya2 as the reference point, the Eya1 gene could not be detected and the Eya3 and Eya4 genes increased about 3 and 8 times, respectively. And is, Eya3 and Eya4 genes except Eya3 ribel gene is significantly decreased in TAC14 did not respond to the effects of the TAC or TAC. Only Eya2 gene increased expression in TAC-R1. Therefore, the present inventors concluded that Eya2 is a physiologically related factor in the regression of cardiac hypertrophy and used Eya2 in subsequent studies. However, the inventors could not exclude the possibility that Eya3 or Eya4 could participate in the cardiac remodeling process in conjunction with Eya2.

We obtained two transgenic mice (TG49 and TG62) using the α-MHC-Eya2 transgene (FIG. 1B). Western blot analysis confirmed that cardiac Eya2 expression was 25 and 5 times higher in TG49 and TG62 than wild type control, respectively (FIG. 1C). Since the two transgenic mice (TG49 and TG62) were indistinguishable in their ability to inhibit the development of cardiac hypertrophy and heart failure (results not shown), we used TG49 mice in subsequent studies. TG49 mice had mild cardiac hypertrophy at baseline compared to WT, such as heart weight / weight (HW / BW) ratio (3.92 ± 0.17, P <0.05 compared to apparent 3.75 ± 0.12) and transverse area of myoblasts ( CSA) (625.1 ± 58.6 μm ² , P <0.01) compared to the apparent 578.3 ± 61.8 μm ² . In addition, as a result of ultrasound cardiac examination, the inventors found that ventricular septal thickness (IVST) (heart expansion: apparent) under reference conditions when the heart without immune response functions (fraction shortening: 47.9 ± 2.68% compared to apparent 48.5 ± 2.38%). 0.92 ± 0.17, P <0.05 compared to 0.63 ± 0.14 mm; cardiac contraction: 1.64 ± 0.18, P <0.05 compared with apparent 1.41 ± 0.18), but a slight but clear increase was observed (FIG. 1D and Table 1).

Ultrasound Heart Check Variations Control Eya2 TG surface TAC 4 weeks TAC 8 weeks TAC 12 weeks surface TAC 4 weeks TAC 8 weeks TAC 12 weeks n = 11 n = 9 n = 6 n = 6 n = 10 n = 12 n = 6 n = 11 BW g 32 ± 3.24 27.5 ± 2.04 ^b 31.0 ± 1.44 31.5 ± 1.73 31.8 ± 1.7 ^d 28.1 ± 3.8 ^b 29.6 ± 1.28 ^c 32 ± 1.75 ^ac IVSTD, mm 0.63 ± 0.14 1.00 ± 0.11 ^a 0.91 ± 0.17 ^a 0.75 ± 0.16 0.92 ± 0.17 ^d 1.00 ± 0.07 1.07 ± 0.17 0.90 ± 0.22 ^a IVSTS, mm 1.41 ± 0.18 1.97 ± 0.10 ^a 1.75 ± 0.22 ^a 1.16 ± 0.15 ^b 1.64 ± 0.18 1.79 ± 0.37 1.87 ± 0.28 1.66 ± 0.36 ^c LVPWD, mm 1.10 ± 0.35 1.14 ± 0.12 1.10 ± 0.33 0.81 ± 0.25 1.34 ± 0.48 ^d 1.45 ± 0.52 1.10 ± 0.23 1.05 ± 0.15 ^d LVPWS, mm 1.46 ± 0.41 1.73 ± 0.34 1.60 ± 0.32 0.96 ± 0.15 ^b 1.64 ± 0.41 1.89 ± 0.49 1.64 ± 0.25 1.38 ± 0.34 ^d LVIDD, mm 3.59 ± 0.21 3.66 ± 0.32 3.36 ± 0.60 3.58 ± 0.27 3.28 ± 0.35 3.25 ± 0.46 ^d 2.92 ± 0.35 3.70 ± 0.27 LVIDS, mm 1.86 ± 0.15 1.66 ± 0.23 ^b 1.70 ± 0.47 2.38 ± 0.30 ^a 1.71 ± 0.21 1.59 ± 0.27 1.32 ± 0.17 ^a 1.96 ± 0.16 ^c EF 85.7 ± 2.10 89.1 ± 4.51 ^b 85.8 ± 5.30 76.9 ± 8.01 ^a 85.2 ± 2.29 87.8 ± 2.75 ^b 89.5 ± 1.98 ^a 84.1 ± 3.86 ^c FS% 48.5 ± 2.38 54.3 ± 7.14 ^b 49.3 ± 6.31 32.8 ± 5.45 ^a 47.9 ± 2.68 51.6 ± 3.58 ^b 54.0 ± 2.94 ^a 47.3 ± 4.65 ^c

Compared to apparent, ^a P <0.01, ^b P <0.05,

^C P <0.01, ^d P <0.05 compared to control (WT)

In addition, markers of cardiac hypertrophy, ANF, BNP and β-MHC, also slightly increased under baseline conditions (FIG. 2C). Thus, Eya2 overexpression does not affect cardiac function under baseline conditions but causes pronounced cardiac hypertrophy, even though mild.

Eya2 Inhibits Cardiac Hypertrophy Induced by Pressure Overload

TG49 and WT mice were treated with TAC for 12 weeks and then structural and functional changes in the heart were monitored. For WT mice, the HW / BW ratio suddenly increased by 47% during the initial 4 weeks of TAC and continued during 12 weeks of TAC compared to the apparently-operated WT mice (eg, 55% at 8 weeks and 65 at 12 weeks). Increase in%). However, in TG49 mice, the HW / BW ratio increased only 31% during the initial 4 weeks of TAC compared to the apparently-operated TG49 mice, and then slightly decreased during the next 8 weeks of TAC (eg, 17% at 8 weeks and 12 weeks). Reduced to 22%). Similarly, the cross-sectional area of myoblasts in WT mice continued to increase during 12 weeks of TAC (eg, 20% at 4 weeks, 54% at 8 weeks, and 91% at 12 weeks), whereas TG49 mice Only increased slightly (eg, 0% at 4 weeks, 23% at 8 weeks and 5% at 12 weeks) (FIG. 2B). Induction of fetal period genes such as ANF, BNP and β-MHC is associated with cardiac hypertrophy. The quantitative RT-PCR experiments confirmed that the induction of these hypertrophic marker genes was clearly reduced in TG49 mice compared to WT mice at 4 weeks of TAC (FIG. 2C). These results indicate that Eya2 inhibits the development of cardiac hypertrophy induced by pressure overload. Interestingly, the expression of α-MHC was induced in TG49 mice while significantly reduced in WT mice (FIG. 2C). Previous reports have shown that the expression of α-MHC is increased in the trained heart [24] and the expression of α-MHC is forced for cardioprotection under conditions of cardiomyopathy [25].

Eya2 Inhibits Heart Failure Induced by Prolonged Pressure Overload

After TAC for 12 weeks, ventricular wall combined with ventricular dilatation (FIG. 3C) (LVIDS, decreased 28% compared to apparent) in WT mice was markedly thinner (FIG. 3A and 3B) (IVSTS, 18% compared to apparent) Reduction; LVPWS, 44% reduction compared to apparent). This structural deformation is associated with a decrease in contractile function (FIG. 3D) (FS: 32% reduction compared to apparent), indicating a switch to heart failure. In contrast, TG49 mice after TAC for 12 weeks showed significantly reduced ventricular wall thinning, ventricular dilatation and contractile muscle abnormality compared to apparently-operated mice (FIG. 3) (IVSTS, no change compared to apparent) ; LVPWS, 16% less than apparent; LVIDS, 15% more than apparent; FS: no change compared to apparent). More detailed echocardiography results are summarized in Table 1.

Heart failure is associated with increased fibrosis and inflammation. Therefore, the heart sections were stained with tricom staining to measure the affected areas (FIG. 4A). Fibrous sites continued to increase under pressure overload in WT mice, whereas very small areas increased in TG49 mice (FIG. 4B). We further confirmed through quantitative RT-PCR that the expression levels of collagen 1, a major component of fibrous resin and TGF-β2, a pro-fibrous cytokine, in TG49 mice were clearly lower compared to WT mice ( 4C). In addition, the induction of the pro-inflammatory cytokine, IL-1β, was prominent in WT mice due to prolonged pressure overload, but significantly decreased in TG49 mice (FIG. 4D). These results confirm that Eya2 inhibits harmful remodeling of the heart, including fibrosis and inflammation.

In conclusion, the data indicate that Eya2 inhibits the development of heart failure.

Eya2 Alters Metabolic Gene Expression and Preserves PI3K / Akt / mTOR Signaling

To understand the molecular mechanisms involving Eya2 in the heart, we performed whole genome microarrays with heart samples from WT and seemingly-operated or aortic stenosis TG49 mice. The data were analyzed by gene set floating analysis (PAGE) [21, 22], which can determine the expression changes accumulated in a series of degenerately defined sets of genes (FIG. 5). First, the gene sets (group 1) affected by TAC in WT mice were determined. These gene sets were compared with altered gene sets in TG49 mice that were either apparently-operated (group 2) or aortic stenosis (group 3). We were able to identify 32 gene sets that reduced expression by TAC in WT but increased expression in apparently-operated or aortic stenosis TG49 mice (FIG. 5A). Of particular interest among the isolated gene sets were those involved in the PPAR signaling pathway, mitochondrial large ribosomal subunit, mitochondrial envelope, fatty acid metabolism and mitochondrial metabolism (FIG. 5B). Pathological cardiac hypertrophy is associated with reduced fatty acid metabolism and physiological cardiac hypertrophy is associated with mitochondrial bioogenesis and increased ability to oxidize fatty acids [26-28]. The results of the present invention thus show the possibility that Eya2 can activate gene programming associated with physiological cardiac hypertrophy. Since the PI3K / Akt / mTOR signal cascade is closely related to motor-induced physiological cardiac hypertrophy [29-32], we measured the activation of the components of the signal cascade using western blotting (FIG. 6A). Densitometric analysis of the blots revealed that the PI3K (p85) level was increased in the heart of TAC49 TAC mice but slightly decreased in the heart of stented WT mice (FIG. 6B). The phosphorylation of p70S6K, a substrate of mTOR, in the heart of TG49 mice was significantly increased (FIG. 6D). These results indicate that the PI3K / Akt / mTOR signal cascade is conserved in the heart of TG49 mice. Further research is needed whether changes in metabolic gene expression and preservation of PI3K / Akt / mTOR signaling are necessary for the beneficial results of Eya2 overexpression.

Additional discussion

Activation of various transcription factors, such as nuclear factor (NFAT) [33], GATA-4 [34], myocadine [35] and β-catenin [36] of activated T-cells, mediates hypertrophic growth of myocardium. Eventually, various independent hypertrophic stimuli appear through activation of general transcription factors, which activate the expression of downstream genes responsible for cardiac remodeling. In addition, many voice modulators have been identified that interfere with hypertrophic signaling [5]. For example, GSK-3β inhibits cardiac hypertrophy by phosphorylating NFAT and GATA-4 and translocating them out of the nucleus [37, 38].

It is a controversial issue whether there is an anti-hypertrophic signaling pathway that does not inhibit the hypertrophic signaling pathway that is activated but leads to a unique cardiac remodeling process as opposed to hypertrophy. In connection with this problem, we isolated gene sets that specifically increase expression during the course of cardiac hypertrophy by implementing gene expression profiles in animal models of hypertrophic degeneration. We deduced that genes that specifically increase expression during the retrograde phase but which do not express during the onset or development of cardiac hypertrophy will be included in the anti- hypertrophic signaling pathway.

We found that Eya2 is a very potent candidate for hypothetical anti-hypertrophic signaling pathway because of increased expression of Eya2 during the degenerative phase and overexpression of Eya2 inhibits PE-induced myocardial cell hypertrophy [6]. In the present invention, we have found that Eya2 inhibits the development of cardiac hypertrophy and heart failure at the molecular, cellular and functional levels. The molecular mechanisms involving Eya2 function in cardiac remodeling are unknown. We propose the possibility that Eya2 triggers physiological cardiac hypertrophy signaling instead of blocking pathological cardiac hypertrophy signaling based on the following two findings: 1) Mytochondrial biogenesis associated with physiological hypertrophy And metabolic gene sets increased expression in Eya2 transgenic mice but markedly decreased in aortic-stenosised WT controls, and 2) the PI3K / Akt / mTOR signal cascade that mediates physiological cardiac hypertrophy was not a WT control It is preserved in mice. Perhaps these quantitative changes in Eya2 transgenic mice in the context of prolonged pressure overload work in a clearly beneficial manner.

It expresses closely related Eya1 and Eya2 in cranial placode, gill arch, central nervous system and developing eye [13]. Eya2 ^{− / −} mice did not show any apparent developmental or morphological defects, whereas Eya1 ^{− / −} mice were embryonic-lethal. Eya1 ^-/- Bone muscle development is temporarily delayed in the embryo. The loss of an allele of Eya2 in the Eya1 ^-/- Eya2 ^+/- fetus results in severe and selective limb muscle hypoplasia, in which Eya1 and Eya2 have redundant functions during muscle development in the mouse fetus. It means that it is [39]. In addition, the inventors have found that overexpression of Eya1 or Eya3 inhibits PE-induced hypertrophy in neonatal cardiomyocytes (not shown results). This means that there is at least functional redundancy between Eya proteins in the overexpressed state. Thus the absence of any cardiac defects in the Eya2 ^{− / −} fetus probably indicates that Eya proteins are not included in early cardiomyogenic or other Eya genes can compensate for Eya2 deficiency. Eya1 can detect expression in heart by RT-PCR showed no expression of Eya2 relatively lower than Eya3 and Eya4. Unlike Eya3 and Eya4, expression of Eya2 is increased by over a period of degeneration hypercardia This means that Eya2 is most closely associated element physiologically in the process. Through aortic stenosis Eya2 ^{− / −} mice, we will be able to determine whether only Eya2 is critically involved in cardiac remodeling or whether Eya3 or Eya4 can compensate for Eya2 deficiency. Human mutations in Eya4 cause hearing loss in perceptual organs and are often accompanied by late-onset dilated cardiomyopathy [18]. Eya4 ^-/- mice have severe hearing impairment and develop into otitis media with effusion [40]. However, no defects in heart morphology or function were reported in Eya4 ^{− / −} mice. Zebrafish embryos infused with morpholino against Eya4 give rise to phenotypes with cardiovascular abnormalities [18]. One of the downstream genes regulated by Eya4 is atp1b2b , which encodes the β2b subunit of Na ⁺ / K ⁺ -ATPase. Na ⁺ / K ⁺ -ATPase is important for the development of the sensory system and maintenance of cardiac function in zebrafish [41]. All phenotypic consequences caused by Eya4 deficiency suggest functional redundancy between Eya2 and Eya4 in the heart. Research is underway on this hypothesis.

Eya proteins are not only transcription factors, but also function as protein tyrosine phosphatase. Destruction of the phosphatase activity severely affects Eya's ability to promote eye formation in Drosophila [14-16]. However, the inventors have ^observed that Eya2 ^D268A , an Eya2 mutant that is expected to disrupt phosphatase activity, can still inhibit PE-induced myocardial cell hypertrophy (not shown). This means that the phosphatase activity of Eya2 is not important for the function involved in cardiac remodeling. Previously, destruction of phosphatase activity did not inhibit Drosophila Eya's ability to function as a transcription factor [16].

In conclusion, the inventors confirmed that Eya2 maintains a compensatory function against complementary cardiac hypertrophy and also inhibits functional and morphological detriment under prolonged pressure overload. Subsequent work will reveal molecular mechanisms involving Eya2 function in the heart. We propose that Eya2 provides a novel therapeutic target for treating cardiac hypertrophy and heart failure.

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Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1 shows the preparation of Eya2 transgenic mice. (A) ANF (n = 3), Eya2 (n = 2), measured semi-sequentially after 14 days of transverse aortic stenosis (TAC14) or after 14 days of TAC without stenosis and after one day (TAC-R1), It is a graph indicating the amount of Eya3 (n = 3) and Eya4 (n = 5) transcripts. (B) Schematic showing Eya2 expression under the control of the α-MHC promoter. (C) Eya2 Western blotting results in cardiac extracts of wild-type control (WT) and Eya2 transformation lines (TG62 and TG49). (D) Heart weight / weight (HW / BW) ratio (WT; n = 9, TG49; n = 7) at cardiac dilatation and heart contraction (WT; n = 11, TG49; n = 10) It is a graph measuring the cross-sectional area (CSA) (n = 3 for both WT and TG49) and the ventricular septum thickness (IVST). ^* P <0.05, ^** P <0.01.

2 is a result showing that Eya2 blocks cardiac hypertrophy. (A) Cardiac cross-section and HW / BW ratio were measured in TAC WT (n = 4-9) and TG49 (n = 7-8) for 12 weeks. (B) Schematic photomicrographs of heart slices showing representative myoblasts. CSA was measured using AnalySIS image analysis program (WT; n = 3-5, TG49; n = 3-4). (C) Graph showing quantitative RT-PCR analysis (n = 3) for ANF, BNP, β-MHC, α-MHC transcripts from heart of TAC mice for 4 weeks, compared to WT ^* P <0.05 , ^** P <0.01, ^Ψ P <0.05.

3 is a result showing that Eya2 inhibits heart failure. (A) Cardiac structure and its function in WT (n = 6-11) and TG49 (n = 6-12) mice TACs for 12 weeks were monitored by ultrasound cardiac examination. Ventricular septal thickness (IVSTS), left ventricular posterior wall thickness (LVPWS), left ventricular internal area (LVIDS) and fractional shortening (FS) values at heart contraction were presented. ^* P <0.05 compared to baseline, ^* P <0.01 compared to baseline, ^Ψ P <0.05 compared to WT.

4 is a result showing that Eya2 inhibits fibrosis and inflammation. (A) Tricom staining was performed on histological sections prepared from the heart in TAC WT and TG49 mice for 12 weeks. (B) The fibrous sites on histological sections are quantified using an AnalySIS image analyzer. Collagen 1, TGF-β2 (C) and IL-1β (D) transcript levels in the heart of TAC mice over 12 weeks are analyzed by quantitative RT-PCR. N = 3 for all groups. ^* P <0.05, ^** P <0.01.

5 is a result of analyzing the gene expression profiles of WT and TG49 by the gene set floating analysis method (PAGE). (A) WT mice (groups) compared to a set of genes that increase or decrease expression in sham-operated (group 2, n = 4) or TAC (group 3, n = 4) TG49 mice 1, n- = 5) sets of genes whose expression is increased (red) or decreased (green) by TAC. (B) Gene sets with reduced expression by TAC in WT mice but increased expression in TG49 mice.

6 shows the PI3K / Akt / mTOR signal cascade activated in TG49 mice. (A) Heart extracts extracted from WT mice or TG49 mice that were apparently-operated or aortically constricted for 12 weeks were isolated by blotting with SDS-PAGE and probed with antibodies. Tubulin was used as a loading control. Blots were scanned and quantified for PI3K (p85) (B), Akt and phospho-Akt (ser473) (C) and p70S6K and phospho-pp70S6K (D) using an AnalySIS image analyzer. N = 3 for all groups. ^* P <0.05, ^** P <0.01.

Claims (10)

Screening methods for preventing or treating heart failure, fibrosis or inflammation comprising the following steps: (a) treating a test substance to a cell comprising an Eya2 (eyes absent 2) gene; And (b) analyzing the expression of the Eya2 gene, and when the test substance increases the expression of the Eya2 gene, it is determined as a prophylactic or therapeutic agent for heart failure, fibrosis or inflammation. The method of claim 1, wherein the fibrosis or inflammation is fibrosis or inflammation in the heart. 3. The method of claim 2, wherein said fibrosis or inflammation causes heart failure. The method of claim 1, wherein the cell is a cell derived from a heart. The method of claim 1, wherein the analysis of gene expression in step (b) is performed by measuring RNA or Eya2 protein of Eya2 gene. The method of claim 1, wherein the method further comprises expression analysis of genes involved in the PI3K / Akt / mTOR signaling pathway. The method of claim 6, wherein the expression analysis of genes involved in the PI3K / Akt / mTOR signaling pathway is performed by detecting PI3K, pAKT, or pp70S6K proteins. A composition for preventing or treating heart failure, fibrosis or inflammation, comprising Eya2 (eyes absent 2) gene as an active ingredient. The composition of claim 8, wherein the fibrosis or inflammation is fibrosis or inflammation in the heart. 10. The composition of claim 9, wherein the fibrosis or inflammation causes heart failure.

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