Use of prmt7 protein or gene expressing same for preventing or treating heart disease

Abstract

The present invention relates to a use of a Prmt7 protein, a gene encoding the Prmt7 protein, or analogs thereof, or a substance regulating the expression of the protein or the gene encoding same for preventing, alleviating, or treating heart disease or protecting endothelial cells.

Description

The use of the PRMT7 protein or the gene expressing it for the prevention or treatment of heart disease

[0001] The present invention relates to the prevention, improvement, or treatment of heart disease or endothelial cell protection through the regulation of the Prmt7 protein or a gene for expressing the same.

[0002] Myocardial infarction (MI) induces left ventricular remodeling and is one of the leading causes of death worldwide. Myocardial infarction is generally triggered by the rupture of coronary artery plaques, which can occur when oxygen supply to the myocardium is insufficient due to myocardial cell death, acute inflammation, angiogenesis, and fibrosis formation. Endothelial cells play a pivotal role in restoring cardiac function after myocardial infarction through vascular recanalization and improved perfusion of the infarct site. Furthermore, endothelial cells contribute to tissue repair by aggregating recovery cells and regulating the remodeling of the extracellular matrix, which is crucial for inflammation and cardiac function recovery. Reducing the size of the infarction by enhancing vascular regeneration is the most effective response to myocardial infarction, and angiogenesis is the primary mechanism for regenerating blood vessels in the infarcted heart. Recently, it has been revealed that stimulation of the proliferative and motile phenotypes of endothelial cells is essential for angiogenesis. Incomplete regeneration of the infarct site can lead to the accumulation of fibrous tissue, reduced barrier function, and microvascular dysfunction associated with permeability and inflammation, ultimately resulting in impaired angiogenesis. Consequently, significant efforts are currently focused on identifying endothelial cells that induce vascular homeostasis and angiogenesis in the infarct site, as well as elucidating their regulatory mechanisms.

[0003] Under various cellular stresses, endoplasmic reticulum (ER) stress signaling plays a crucial role in controlling cell survival and death and is associated with cardiovascular homeostasis and the onset of disease. Ischemic injury, such as myocardial infarction, increases the modulated homeostatic function of the ER, which is activated through distinct pathways via the unfolded protein response (UPR). Phosphorylation of eukaryotic initiation factors 2alpha (eIF2α) by endoplasmic reticulum kinase (PRKR-like endoplasmic reticulum kinase, PERK) induces overall inhibition of translation and promotes the translation of specific proteins, such as activating transcription factor 4 (ATF4), which are involved in protein folding, oxidative stress, and amino acid metabolism. Under sustained ER stress conditions, the expression of CCAAT / enhancer-binding homologous protein (CHOP) by ATF4 initiates apoptosis-promoting pathways. Several studies have reported the role of ER stress in angiogenesis and endothelial dysfunction through the regulation of cell proliferation and apoptosis. In contrast to the induction of endothelial cell apoptosis caused by uncontrolled or prolonged ER stress, vascular endothelial growth factor (VEGF) signaling contributes to angiogenesis by activating cell survival signals through transient ER stress and high levels of AKT phosphorylation activation. However, the detailed molecular mechanisms that balance endothelial cell apoptosis and proliferation in response to ischemic injury have not been fully elucidated.

[0004] Protein arginine methyltransferases (Prmts) regulate signaling pathways and gene expression by methylating histone and non-histone substrates for arginine residues. Various Prmt proteins have been reported to be involved in cardiovascular function and disease. For example, it has been reported that inhibiting the expression of Prmt5 in endothelial cells can impede post-ischemic recovery by attenuating VEGF signaling and inducing angiogenesis, but similar effects on other Prmts are still under investigation.

[0005]

[0006] The inventors have discovered that Prmt7dl plays an important role in the survival and proliferation of endothelial cells and protects endothelial cells from cell death induced by myocardial cell death, acute inflammation, angiogenesis, and fibrosis formation, thereby having an effect on the prevention or treatment of heart disease, and furthermore, ischemic heart diseases such as myocardial infarction or ischemic cardiomyopathy, and have completed the present invention.

[0007] Accordingly, the object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of heart disease comprising: a Prmt7 protein; a gene encoding said Prmt7 protein or an analog thereof; or a preparation that promotes the expression or activity of the gene encoding said Prmt7 protein.

[0008] Another object of the present invention is to provide a health functional food composition for the prevention or improvement of heart disease comprising: a Prmt7 protein; a gene encoding said Prmt7 protein or an analog thereof; or a preparation that promotes the expression or activity of the gene encoding said Prmt7 protein.

[0009] Another object of the present invention is to provide a health functional food composition for protecting endothelial cells (ECs), comprising: a Prmt7 protein; a gene encoding said Prmt7 protein or an analog thereof; or a preparation that promotes the expression or activity of the gene encoding said Prmt7 protein.

[0010] Another objective of the present invention is to provide a screening method for therapeutic substances for heart disease, comprising the steps of: treating endothelial cells with a candidate substance; measuring the expression or activity of a Prmt7 protein in the endothelial cells treated with the candidate substance; and selecting a substance in which the activity or expression of the Prmt7 protein is increased compared to a control group not treated with the candidate substance as a therapeutic substance for ischemic heart disease.

[0011]

[0012] To achieve the above objectives, the present invention provides a pharmaceutical composition for the prevention or treatment of heart disease comprising: a Prmt7 protein; a gene encoding the Prmt7 protein or an analog thereof; or a preparation that promotes the expression or activity of the gene encoding the Prmt7 protein.

[0013] To achieve another objective of the present invention, the present invention provides a health functional food composition for the prevention or improvement of heart disease comprising: a Prmt7 protein; a gene encoding the Prmt7 protein or an analog thereof; or a preparation that promotes the expression or activity of the gene encoding the Prmt7 protein.

[0014] To achieve another objective of the present invention, the present invention provides a health functional food composition for protecting endothelial cells (EC), comprising: a Prmt7 protein; a gene encoding said Prmt7 protein or an analog thereof; or a preparation that promotes the expression or activity of the gene encoding said Prmt7 protein.

[0015] To achieve another objective of the present invention, the present invention provides a method for screening therapeutic substances for heart disease, comprising the steps of: treating endothelial cells with a candidate substance; measuring the expression or activity of a Prmt7 protein in the endothelial cells treated with the candidate substance; and selecting a substance in which the activity or expression of the Prmt7 protein is increased or increased compared to a control group not treated with the candidate substance as a therapeutic substance for ischemic heart disease.

[0016]

[0017] The present invention will be described in detail below.

[0018] In one aspect of the present invention, the present invention relates to a pharmaceutical composition for the prevention or treatment of heart disease comprising: a Prmt7 protein; a gene encoding said Prmt7 protein or an analog thereof; or a preparation that promotes the expression or activity of the gene encoding said Prmt7 protein.

[0019] The Prmt7 (Protein arginine methyltransferase 7) of the present invention is an enzyme that mediates methylation of arginine residues on histones and non-histones, and can regulate various biological signaling pathways. In particular, in the present invention, Prmt7 plays an important role in inhibiting the apoptotic response to endoplasmic reticulum (ER) stress arising from ischemic heart disease and in activating cell survival signals, and can perform a role in protecting cardiac function. When ER stress persists, protein translation is inhibited due to the phosphorylation of eIF2α by PERK, and the expression of CHOP by ATF4 initiates the pro-apoptotic pathway. That is, ER stress can promote damage or death of endothelial cells, which can also occur due to ischemic damage.

[0020] In addition, the present invention may include an analogue of the Prmt7 protein or a gene encoding it, and the analogue of the Prmt7 protein may be a protein or peptide that performs the same function as Prmt7, or may include a peptide in which a specific residue that does not affect the function is modified. In addition, the gene encoding the Prmt7 protein is not limited to the form of DNA, RNA, mRNA, etc., and may include an analogue in which a portion of the sugar or base of the gene is modified, or may include a nucleic acid sequence modified through the addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0021] The Prmt7 of the present invention is primarily expressed in endothelial cells of the coronary arteries and plays a role in regulating ER (endoplasmic reticulum) stress. That is, Prmt7 promotes the inhibition of apoptosis caused by ER stress and the activation of cell survival signals in endothelial cells of coronary arteries, and consequently promotes the proliferation of endothelial cells. In addition, through this, it can exhibit a preventive or therapeutic effect against ischemic heart diseases such as myocardial infarction.

[0022] In the present invention, the term "heart disease" refers to a disease caused by the inhibition of endothelial cell proliferation or death, and specifically refers to a heart disease caused by various ER stresses. In particular, the heart disease may be an ischemic heart disease, and "ischemic heart disease" refers to a heart disease that may be caused by the narrowing or blockage of coronary arteries due to various causes such as damage to endothelial cells, inflammation, or death of myocardial cells. Specifically, the ischemic heart disease may include myocardial infarction, ischemic cardiomyopathy, myocardial ischemia, and angina pectoris, and preferably may be myocardial infarction or ischemic cardiomyopathy.

[0023] In one embodiment of the present invention, an experiment was conducted to determine whether Prmt7 affects the protection of coronary artery endothelial cells (ECs), that is, the proliferation and growth of ECs. Specifically, it was confirmed that in animal models induced with ischemic heart disease such as myocardial infarction (MI), ischemic cardiomyopathy, myocardial ischemia, and angina pectoris, the expression or activation of Prmt7 can have the effect of preventing and treating said diseases or alleviating cardiac dysfunction caused by them.

[0024] That is, the present invention is a pharmaceutical composition comprising a Prmt7 protein or a nucleic acid molecule expressing the Prmt7 protein, wherein the pharmaceutical composition has a preventive or therapeutic effect on heart disease, more specifically, ischemic heart disease such as myocardial infarction, ischemic cardiomyopathy, myocardial ischemia, and angina pectoris, or heart disease including fibrosis.

[0025] In this specification, the term "prevention" refers to any act that can suppress heart disease or delay its onset by administering the pharmaceutical composition according to the present invention.

[0026] In this specification, the term "treatment" refers to any act in which symptoms related to heart disease are improved or benefited by the administration of the pharmaceutical composition according to the present invention.

[0027] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, and sterile injectable solutions, according to conventional methods, and may additionally include carriers or excipients necessary for the formulation. Pharmaceutically acceptable carriers, excipients, and diluents that may additionally be included in the active ingredient include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, and mineral oil. When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0028] For example, solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as cotton, starch, calcium carbonate, sucrose or lactose, or gelatin, with the extract or compound. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and may include various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0029] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, witepsol, macrogol, tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used.

[0030] The pharmaceutical composition of the present invention may be administered orally or parenterally (intravenous injection, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage may vary depending on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the time, and may be selected in an appropriate form by those skilled in the art.

[0031] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceuticalally effective amount" refers to a reasonable amount applicable to medical treatment, meaning an amount sufficient to treat a disease, and the criteria may be determined based on the patient's disease, severity, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concomitant components, and other factors. The pharmaceutical composition of the present invention may be administered in combination with an individual therapeutic agent or other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above factors, the dosage may be determined to a level that minimizes side effects, and this can be easily determined by a person skilled in the art. Specifically, the dosage of the pharmaceutical composition may vary depending on the patient's age, weight, severity, gender, etc., and generally, an amount of 0.001 to 150 mg, more preferably 0.01 to 100 mg per kg of body weight, may be administered daily or every other day, 1 to 3 times a day. However, this is for illustrative purposes only, and the above dosage may be set differently as needed.

[0032] Furthermore, the present invention relates to a food or health functional food for the prevention or improvement of heart disease comprising Prmt7 or a nucleic acid molecule expressing it. The term "health functional food" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body pursuant to Article 6727 of the Health Functional Foods Act, and the term "functional properties" refers to consuming the food for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0033] The food or health functional food of the present invention can be manufactured and processed into pharmaceutical administration forms such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups, etc., or into health functional foods such as tea bags, infusions, beverages, candies, jellies, and gums for the purpose of preventing and improving ischemic heart disease.

[0034] The food or health functional food composition of the present invention may be used as a food additive and may be manufactured into a product either alone or in combination with other ingredients. Additionally, it may include nutritional supplements, vitamins, electrolytes, flavoring agents, coloring agents and promoters, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. The above ingredients may be used alone or in combination, and may be combined and used in appropriate amounts.

[0035] In another aspect of the present invention, the present invention relates to a composition for protecting endothelial cells (EC) comprising a Prmt7 protein or a nucleic acid molecule expressing the Prmt7 protein.

[0036] The term "endothelial cell protection" above refers to enhancing the survival and proliferation of endothelial cells and protecting them from apoptosis. These endothelial cells play a pivotal role in the recovery of cardiac function after myocardial infarction (MI) through vascular recanalization and improved perfusion of the infarct site. Furthermore, endoplasmic reticulum (ER) stress signals play a crucial role in regulating cell survival and death, and Prmt7 of the present invention has been identified as playing a role in regulating these ER stress signals. Endothelial cell protection means improving the function of vascular endothelial cells, inhibiting endothelial cell damage, and reducing oxidative stress induced in endothelial cells.

[0037] In addition, the above-mentioned endothelial cell protective composition can improve vascular function, which may include the effect of improving vascular elasticity or blood flow.

[0038] The endothelial cell protective composition of the present invention may be prepared in the form of a food composition or a food additive, and in particular, may be prepared in the form of a health food composition. The food composition is as described above. Accordingly, the endothelial cell protective composition of the present invention can increase the survival and proliferation of endothelial cells, and furthermore, improve vascular recanalization and infarction reperfusion after myocardial infarction, and influence neovascularization in the relevant area, thereby helping to restore cardiac function.

[0039] In addition, the present invention relates to a screening method for a therapeutic substance for heart disease comprising: 1) treating endothelial cells with a candidate substance; 2) measuring the activity of the Prmt7 protein or its gene in the endothelial cells treated with the candidate substance; and 3) selecting a substance in which the activity of the Prmt7 protein is increased or the expression level is increased compared to a control group not treated with the candidate substance as a therapeutic substance for heart disease.

[0040] In the present invention, the candidate substance is a substance that regulates the expression or activity of the Prmt7 protein or the gene encoding it, and is not limited to any type such as natural products, compounds, peptides, or nucleic acid molecules; any substance that generally enhances or promotes the expression or activity of the Prmt7 protein or the gene encoding it in order to be included in the composition of the present invention may be used without limitation.

[0041] In addition, the step of measuring the expression or activity of the above-mentioned Prmt7 protein or its gene may be carried out through various methods capable of measuring the amount of RNA or protein produced by the expression of the Prmt7 gene. Preferably, the amount of RNA expression may be confirmed using known methods such as RT-PCR (real-time PCR), microarray, and Northern blotting, and the activity and expression of the protein may be confirmed using known methods such as immunoprecipitation, immunostaining, chromatin immunostaining, enzyme immunoassay (ELISA), and Western blotting, but are not limited thereto.

[0042] Furthermore, the present invention relates to an animal model that increases the occurrence or progression of heart disease by specifically inducing a decrease in the expression of the Prmt7 protein or the gene encoding said Prmt7 protein in heart cells.

[0043] The animal model according to the present invention consists of individuals in which the Prmt7 protein is genetically knocked out or its expression is reduced. These individuals with reduced Prmt7 protein have the characteristic of having an increased incidence or progression of heart disease, particularly ischemic heart disease selected from myocardial infarction (MI), ischemic cardiomyopathy, myocardial ischemia, and angina pectoris, compared to normal individuals.

[0044] The above animal models are not limited to any specific type and may include mammals such as mice, rats, rabbits, guinea pigs, hamsters, and ferrets. The above animal models may be used for the purpose of screening therapeutic candidate substances for heart disease caused by Prmt7 deficiency or for evaluating the pharmacological effects of said substances.

[0045]

[0046] The present invention relates to the prevention, improvement, or treatment of heart disease or endothelial cell protection through the regulation of the Prmt7 protein, the gene encoding the Prmt7 protein, or an analog thereof. Since PRMT7 enhances the survival and proliferation of heart endothelial cells and further inhibits apoptosis, it can have excellent preventive or therapeutic effects against heart disease such as myocardial infarction through its direct expression or regulation of expression.

[0047]

[0048] Figure 1 shows the results of confirming changes in the expression level of Prmt7 in coronary artery endothelial cells and various types of heart cells of mice with induced myocardial infarction.

[0049] Figure 1a shows the types and locations of cells in which Prmt7 is expressed, confirming that it is expressed at high levels in endothelial cells, epicardial cells, macrophages, and fibroblasts, whereas it is expressed at relatively low levels in Schwann cells, B cells, neutrophils, and smooth muscle.

[0050] Figure 1b shows the results of confirming the amount of Prmt7 expressed from heart cells after 1 week or 3 weeks, respectively, following the induction of myocardial infarction (MI).

[0051] Figure 1c shows the results of comparing the expression levels of Prmt1 and related factors in the heart 3 weeks after inducing myocardial infarction (MI).

[0052] Figure 1d shows the results of comparing the expression levels of myocardial infarction-related factors (Vcam, Icam, TGF-β1, IL-1α) expressed in the heart 3 weeks after inducing myocardial infarction (MI).

[0053] Figure 1e is a fluorescent staining image confirming the expression level of Prmt7 in major heart cells.

[0054] Figure 2 shows the effect of endothelial cell-specific Prmt7 deficiency on myocardial infarction-induced cardiac dysfunction, and Figure 2a is a schematic diagram of the experimental process to confirm this.

[0055] Figure 2b shows the results of measuring heart rate in an animal model after 1 and 3 weeks following the induction of myocardial infarction (MI).

[0056] Figures 2c and 2d show the results of echocardiography analysis for the endothelial cell-specific Prmt7 deficiency-myocardial infarction-induced model (EndoKO-MI), the myocardial infarction-induced model (MI), and the control group (sham).

[0057] Figure 2e shows the results of measuring left ventricular weight for the endothelial cell-specific Prmt7 deficiency-myocardial infarction-induced model (EndoKO-MI), the myocardial infarction-induced model (MI), and the control group (sham).

[0058] Figure 2f shows the results of measuring the aortic acceleration time for each group, and Figure 2g shows the results of measuring the pulmonary vascular velocity time (PV VTI), pulmonary acceleration time (PAT), and pulmonary extraction time (PET), respectively.

[0059] Figure 3 shows the results of confirming the progression of cardiomyopathy and fibrosis in mice in which Prmt7 was knocked out in endothelial cells.

[0060] Figures 3a and 3b show the results of staining with EB to check for cardiovascular leakage in EndoKO-MI, MI, and the control group (WT).

[0061] Figures 3c and 3d show the results of tissue staining using H&E and MT in EndoKO-MI, MI, and control (WT).

[0062] Figure 3e shows the results of confirming the cross-sectional area of ​​myocardial cells for each group, and Figures 3f and 3g show the results of confirming cell death for each group.

[0063] Figure 3h shows the results of comparing the expression status and expression amount of cell stress markers for each group.

[0064] Figure 3i shows the results of identifying the location of connexin 43, a component of intercellular gap junction proteins, in each group.

[0065] Figure 4 shows the results of confirming the effects of Prmt7 on endothelial cell proliferation and apoptosis.

[0066] Figure 4a shows the results of reduced cell viability when endothelial cells were treated with SGC8158 to inhibit Prmt7 activity, and Figures 4b and 4c show the results confirmed through fluorescent staining.

[0067] Figure 4d shows the change in expression levels of apoptosis regulator (Bax) and proliferation marker (Ki67) when endothelial cells are treated with SGC8158 to inhibit Prmt7 activity.

[0068] Figure 5 confirms the effect on ER stress-mediated endothelial cell damage upon Prmt7 inhibition, and shows the results of confirming whether the expression of various cell damage factors and apoptosis regulators changes depending on Prmt7 inhibition.

[0069]

[0070] Hereinafter, embodiments are described in detail to specifically explain the present specification. However, the embodiments according to the present specification may be modified in various different forms, and the scope of the present specification is not to be interpreted as being limited to the embodiments described below. The embodiments of the present specification are provided to more completely explain the present specification to those with average knowledge in the art.

[0071]

[0072] Experimental Methods and Materials

[0073] 1. Preparation of animal models for mouse experiments

[0074] Prmt7 mice were purchased from the Sanger Institute, and Prmt7± mice were prepared by backcrossing on a C57BL / 6 J background for at least 10 generations. In all experiments, Prmt7 - / - Littermate wild-type mice were used as the control group, and Prmt7 was used to construct heart-specific Prmt7 null mice. Tm1c / Tm1c (Prmt7 f / f Prmt7 was crossed with mice carrying the VE-cadherin-CreERT2 transgene (Jackson Laboratory; Tg(Cdh5-cre)7Mlia / J). To evaluate the effect of Prmt7 on CVD, approximately 3-month-old male mice from a heterozygous breeding litter were used in this study. Additionally, LAD ligation was performed on 10-week-old mice to induce myocardial infarction (MI). Animal studies were conducted in accordance with ethical guidelines (protocol number: SKKUUIACUC2023-02-21-1) with the approval of the Institutional Animal Care and Use Committee (IACUC) of Sungkyunkwan University School of Medicine (SUSM).

[0075] In addition, a mouse model of myocardial infarction (MI) was established via left coronary artery (LCA) ligation without mechanical circulation, as in the known method (Gao et al., 2010, Cir Res). Specifically, mice were anesthetized with 2% isoflurane inhalation without ventilation, a 0.5 cm skin incision was made on the left chest, and a sac-like suture was created. The hepatic cutaneous muscle was exposed through venous incisions in the skull and pleura of both chests. Then, the mouse heart was removed by compressing the right chest wall, the LCA was ligated, and the heart was immediately returned to the chest and sutured. The control group underwent the same surgical procedure without ligating the LCA.

[0076]

[0077] 2. Echocardiogram Analysis

[0078] After anesthetizing mice with 1% (vol / vol) isoflurane, echocardiography was performed on 3-month-old mice and 1 day before sacrifice using a Vevo LAZR-X photoacoustic imaging system (Fujifilm Visual Sonics). Heart rate was monitored and generally maintained at 400–500 beats per minute. M-mode image analysis derived from the short-shortened view of the left ventricle (LV) was performed to calculate ejection fraction (EF) and short-shortened fraction (FS).

[0079]

[0080] 3. Histology and Immunohistochemistry

[0081] Histology of cardiac sections was performed according to known methods. Excised mouse hearts were fixed in 4% paraformaldehyde (PFA) and stored in paraffin blocks at the optimal cutting temperature (OCT, Sakura Finetec). Paraffin-containing cardiac tissues were sectioned to a thickness of 7 μm and stained with hematoxylin and eosin (H&E, BBC Biochemical) and Masson's trichrome (Abcam). Immunohistochemistry of Prmt7 in cardiac samples was performed according to previously known methods. The stained cardiac tissues were sectioned to a thickness of 7 μm and subjected to sequential rehydration and antigen recovery in 0.15% PBS buffer with a primary antibody diluted 1:200 in 2% (wt / vol) BSA solution. Images were analyzed using a Cytation C10 fluorescence microscopy system (BioTek).

[0082]

[0083] 4. Evans blue staining

[0084] Evans Blue staining was used to analyze apoptosis within the myocardium. Evans Blue (Sigma-Aldrich) was dissolved in PBS (10 mg / ml) and sterilized by passing it through a membrane filter with a pore size of 0.2 μm. The Evans Blue solution was then injected into mice via the tail vein at a concentration of 1% w / v. Three hours after injection, the mice were sacrificed, and staining into the myocardium was visually confirmed.

[0085]

[0086] 5. TUNEL Staining

[0087] TUNEL imaging (Click-iT plus TUNEL Assay) was performed to investigate cardiac apoptosis. After penetration, cardiac sections were attached with a mixture of longitudinal deoxynucleotide transferase buffer and EdUTP. After 60 minutes of inkation, the sections were treated in 3% BSA, 0.1% Triton X-100, PBS for 5 minutes and mounted with a mounting solution containing DAPI (Abcam) to visualize the nuclei. Images were analyzed using a Cytation C10 fluorescence microscopy system (BioTek).

[0088]

[0089] 6. Cell Culture

[0090] Endothelial cells (EC, C166) were cultured in Dulbecco's Modified Eagle's Medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin / streptomycin under standard culture conditions (37°C and 5% CO2). To evaluate the inhibitory effect of Prmt7 under stress conditions, cells were treated with 1 μM SGC8158, a Prmt7 inhibitor (Sigma-Aldrich), along with 0.2% BSA or 50 ng / ml TNFα (PeproTech).

[0091]

[0092] 7. Bromodeoxyuridine (BrdU) staining

[0093] To investigate cell proliferation characteristics, C166 cells were grown on chamber slides at appropriate confluence points. After treatment with various conditions, cells were cultured in fresh medium containing BrdU at 10 μM for 4 hours, fixed in cold methanol (-20°C) at 4°C for 4 minutes, and then hydrochloride denaturation was performed at 2 M for at least 20 minutes at room temperature. Cells were blocked with 3% BSA in PBS at room temperature for 1 hour, treated with the primary anti-BrdU antibody and the secondary antibody Alexa Fluor 546, and then mounted using a mounting solution containing DAPI (Abcam). Images were analyzed using a Cytation C10 confocal microscope system (BioTek).

[0094]

[0095] 8. Scratch Analysis

[0096] To investigate migration ability, 5×10 C166 cells 4 Cells were seeded into a 6-well plate at a density of approximately 90%. A pipette tip was used to create mechanical scratches in the cell monolayer. Images were taken after various time intervals (0, 12, 24, 36, and 48 hours), and migration characteristics were quantified through the wound closure rate.

[0097]

[0098] 9. Protein Analysis

[0099] Immunoblotting analysis was performed in a known manner. Briefly, lysed tissues or cultured cells were lysed using a lysis buffer composed of 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and a complete protease inhibitor mixture (Roche Diagnostics). Protein concentrations were measured at 562 nm using a spectrophotometer with a BCA protein assay reagent (Pierce). Cell lysates were applied to SDS-PAGE (Bio-Rad) and transferred to a PVDF membrane (Bio-Rad). The membrane was blocked with Tris-buffered saline (TBST) and 5% bovine serum albumin for 30 minutes and incubated overnight at 4°C with the indicated primary antibodies. The blot was washed with TBST for 20 minutes, incubated with a peroxidase-conjugated secondary antibody at room temperature for 1.5 hours, washed with TBST for 20 minutes, and visualized. Protein-level quantification was obtained by signal intensity analysis using the Image J (NIH) program and normalized to a loading control.

[0100]

[0101] 10. RNA Analysis

[0102] qRT-PCR and RNA sequencing analysis were performed according to known methods. Total RNA from mouse heart and C166 cells was extracted using easy-BLUE (iNtRON) reagents according to the manufacturer's instructions. Primer sequences for qRT-PCR are listed in Table 1 below. cDNA samples were generated from 0.5 μg of RNA using the PrimeScript RT reagent kit (TaKaRa) according to the manufacturer's protocol. RNA sequencing analysis was performed using an Agilent 2100 bioanalyzer with an RNA 6000 Nano Chip (Agilent Technologies). Analysis of RNA sequencing data was performed using ExDEGA v1.61 (e-Biogen) and displayed with MeV (v4.9.0) software. Total gene expression was evaluated by reacting with the MSigDB database v6.1 (>1.3-fold, RC log2>2, P<0.05) using Gene Set Enrichment Analysis (http: / software.broadinstitute.org).

[0103] Gene symbol 5' to 3' Sequence number Prmt7 Forward5'-TTC-CCA-CAG-CGG-GCA-TTA-T-3' Sequence number 1 Reverse5'-TGT-AGC-ATG-TCG-GCA-TAG-GA-3' Sequence number 2 Ki67 Forward5'-TCA-TGA-GGA-TGG-AAG-CAA-GCC-3' Sequence number 3 Reverse5'-CTC-ACT-CTT-CTC-AGG-GTC-AGC-A-3' Sequence number 4 Vcam Forward5'-GCC-ACC-CTC-ACC-TTA-ATT-GCT-ATG-3' Sequence number 5 Reverse5'-TGT-GCA-GCC-ACC-TGA-GAT-CC-3' Sequence number 6GapdhForward5'-GAC-ATG-CCG-CCT-GGA-GAA-AC-3'Sequence No. 7Reverse5'-AGC-CCA-GGA-TGC-CCT-TTA-GT-3'Sequence No. 8SOD2Forward5'-TTA-ACG-CGC-AGA-TCA-TGC-A-3'Sequence No. 9Reverse5'-GGT-GGC-GTT-GAG-ATT-GTT-CA-3'Sequence No. 10IL-1αForward5'-GGA-GAA-GAC-CAG-CCC-GTG-TTG-CT-3'Sequence No. 11Reverse5'-CCG-TGC-CAG-GTG-CAC-CCG-ACT-T-3'Sequence No. 12IL6Forward5'-CAA-CGA-TGA-TGC-ACT-TGC-AGA-3'Sequence No. 13Reverse5'-GTG-ACT-CCA-GCT-TAT-CTC-TTG-GT-3' Sequence No. 14

[0104] 7. Statistical Analysis

[0105] All experiments were repeated at least three times with identical or similar results. Data represent biological replicates. Appropriate statistical analyses were performed for each analysis. The data satisfied the hypotheses of the statistical tests described for each experiment. The statistical significance of the differences was determined according to GraphPad Prism 8.4.3 (GraphPad Software Inc., San Diego, California, USA), and the P-value * P < 0.05,** P < 0.01, *** P < 0.001, **** P < 0.0001 was considered statistically significant.

[0106]

[0107] Example 1. Changes in Prmt7 expression levels in a myocardial infarction-induced model

[0108] According to the analysis of a published dataset in a mouse model of myocardial infarction (MI), Prmt7 is expressed at high levels in endothelial cells, epicardial cells, granulocytes, and fibroblasts, while showing relatively low expression levels in Schwan cells, B cells, neutrophils, and smooth muscle (Fig. 1a). One week after MI induction, the number of endothelial cells in the heart decreases, while epicardial cells, M1 / M2 granulocytes, and fibroblasts increase. Two weeks after MI induction, endothelial cells and epicardial cells increase significantly in the heart. Three weeks after MI induction, endothelial cells, NK cells, and T cells increase further in the heart, while epicardial cells and granulocytes decrease. Prmt7 is expressed most highly in cardiac cells one week after MI induction (Fig. 1b).

[0109] In addition, Prmt7 mRNA and protein levels were significantly reduced in mouse hearts 3 weeks after MI induction, while cleaved-caspase 3 (c-cas3) and p53 proteins were significantly increased in MI-induced hearts (Fig. 1c). Furthermore, the expression of vascular cell adhesion molecules (Vcam), intercellular adhesion molecules (Icam), TGFα1, and Il-1α showed an increasing trend in MI-induced hearts (Fig. 1d). As with the dataset analysis, Prmt7 was expressed in endothelial cells, smooth muscle cells, and cardiomyocytes, and fluorescent staining images confirmed that Prmt7 expression decreased in these major cardiac cell types after MI induction (Fig. 1e). These data confirm that Prmt7 affects endothelial cells during the early stages of myocardial infarction.

[0110]

[0111] Example 2. Induction of myocardial infarction in a Prmt7 knockout mouse model

[0112] To investigate the effects of Prmt7 on myocardial infarction, electrocardiograms were performed on mouse models in which Prmt7 was knocked out in endothelial cells (EndoKO) and wild-type (WT) mouse models (Figs. 2a, 2b). At MI-1 week, EndoKO-MI mice showed a significant increase in heart rate and standard deviation of the NN interval (SDNN) compared to Sham and WT-MI mice. Both the WT and EndoKO-MI groups showed a significant increase in QRS and QT durations. At MI-3 weeks, the EndoKO-MI group still showed a significant increase in QRS and QT durations, while the WT-MI group showed a slight increase in QT duration. Ultrasound examinations of the Sham and MI groups after 3 weeks showed a significant decrease in ejection fraction (EF) and fraction shortening (FS) in the MI group, whereas cardiac function deteriorated in EndoKO mice (Figs. 2c and 2d).

[0113] Furthermore, EndoKO mice showed an additional reduction in left ventricular mass through remodeling that thinned the left ventricular wall compared to the Sham and WT-MI groups (Fig. 2e). The MI group exhibited hemodynamic changes with a significantly reduced AAT (aortic acceleration time) (Fig. 2f), and significant changes in right ventricular function were observed in the EndoKO-MI group compared to the WT-MI group in evaluations of pulmonary vascular velocity time (PV VTI), pulmonary acceleration time (PAT), and pulmonary extraction time (PET). The WT-MI group showed a typical increase in right ventricular compensatory function (Fig. 2g). Since the increase in compensatory right ventricular contractile function after MI was not observed in the KO group, these data suggest that when Prmt7 is knocked out, it induces myocardial hypertrophic activity, thereby interfering with cardioprotection and potentially worsening cardiac function. In other words, synthesizing the above results, it can be concluded that the removal of Prmt7 from endothelial cells after myocardial infarction may worsen cardiac function.

[0114]

[0115] Example 3. Effects on cardiac disease in Prmt7 knockout mice

[0116] Three weeks after inducing MI, the mouse groups of MI, EndoKO-MI, and WT were stained with Evans Blue (EB) to evaluate vascular leakage (Figure 3a). In the epicardium, large amounts of EB accumulated at the trauma site in the WT-MI and EndoKO-MI hearts, whereas no EB accumulated in the myocardium was observed in the sham-operated hearts. However, in EndoKO-MI, EB was observed more extensively in the myocardium compared to WT-MI. Additionally, compared to the sham group, the WT-MI group showed a relatively slight increase in cardiac mass, while the EndoKO-MI group showed a very large increase in cardiac mass (Figure 3b).

[0117] Furthermore, tissue staining results obtained with hematoxylin-eosin (H&E) and Mason trichrome (MT) showed that while the MI heart exhibited large areas of necrosis and fibrosis, the Enko-MI heart showed a more exacerbated response (Figs. 3c and 3d). As cardiac mass increased, the cross-sectional area of ​​myocardial cells in the EndoKO-MI heart expanded more significantly than in the WT-MI heart, consistent with this (Fig. 3e). TUNEL staining results showed that the EndoKO-MI heart exhibited a significantly increased apoptosis in trauma sites, including the cardiovascular region, compared to the WT-MI heart (Figs. 3f and 3g).

[0118] To confirm these traits, cardiac extracts from the sham, WT-MI, and EndoKO-MI groups were subjected to immunoblotting for fibrosis and cellular stress markers (Figure 3h). As a result, similar to the fibrosis staining, Collagen I and Collagen III were significantly increased in the hearts of the MI group compared to the sham hearts, while EndoKO-MI hearts exhibited much higher levels of these markers, indicating increased fibrosis. Additionally, TGFα levels showed a similar increasing trend in the MI group. Furthermore, myocardial troponin I (cTnI), a marker of cardiac damage, was significantly increased in the MI group, and EndoKO-MI hearts demonstrated a high level of cardiac damage. Moreover, p53 and c-cas3 levels were increased in MI hearts, and it was confirmed that these levels were even higher in EndoKO-MI hearts.

[0119] Gap junctions in myocardial cells are a key mechanism controlling electrical connections for normal cardiac function. Therefore, we investigated the localization of the connexin 43 (Cx43) protein, a key component of gap junction proteins, in ventricular myocardial cells (Figure 3i). Gap junction structure was disrupted in both MI hearts and further deteriorated in EndoKO-MI. Alterations in gap junction structure can lead to cardiac dysfunction. Taken together, these data demonstrate that endothelial Prmt7 knockout in myocardial infarction can exacerbate cardiac stress and mortality.

[0120]

[0121] Example 4. Effects of Prmt7 on Endothelial Cell Proliferation and Cell Survival

[0122] To investigate the role of Prmt7 in endothelial cell proliferation and apoptosis, the effects of Prmt7 inhibition on endothelial cells were examined. C166 endothelial cells were treated for 24 hours with either a vehicle (control) or SGC8158, a Prmt7-specific inhibitor, and cell viability and the uptake of 5-bromo-2'-deoxyuridine (BrdU) were assessed. Compared to cells treated with the control vehicle, cells treated with SGC8158 showed a significantly reduced cell viability (Fig. 4a). Furthermore, inhibition of Prmt7 significantly reduced endothelial cell proliferation compared to the control group (Figs. 4b and 4c). Moreover, Prmt7 inhibition significantly decreased the expression of the proliferation marker Ki67 and increased the expression of Bax (BCL2-associated X), a regulator of apoptosis (Fig. 4d). These data demonstrate that Prmt7 is necessary for the survival and proliferation of endothelial cells.

[0123]

[0124] Example 5. Effect of Prmt7 on ER stress

[0125] Among cellular stresses, ER stress is one of the major factors associated with apoptosis in ischemic heart and endothelial function. The role of Prmt7 in the ER stress response of MI heart and endothelial cells was investigated by considering eIF2α and HSP70 family proteins, which are targets of Prmt7 and are associated with ER stress. Endothelial cells treated with SGC8158 for 24 hours showed increased ER stress and apoptosis, as indicated by elevated levels of gamma-H2AX (a gene damage marker), c-cas3, phosphorylated-eIF2α (p-eIF2α), ATF4, and CHOP proteins compared to the control group (Fig. 5a). Furthermore, inhibition of Prmt7 in endothelial cells increased the level of phosphorylated-p65, confirming that this affects inflammatory responses and ER stress (Fig. 5b).

[0126] Heart extracts from mice 3 weeks after MI induction showed significantly increased levels of phosphorylated-eIF2α (p-eIF2α), ATF4 (activating transcription factor 4), and its target CHOP (C / EBP-like protein) compared to sham hearts (Fig. 5c). These data suggest that ER stress was actually increased simultaneously with the cardiomyocyte death observed in MI hearts.

[0127] Consistent with cardiac function data, the EndoKO-MI group showed higher levels of phosphorylated-eIF2α, ATF4, and CHOP proteins compared to the WT-MI group, demonstrating an exacerbated ER stress response. These data indicate that the ER stress response plays a significant role in ischemia-induced apoptosis, and confirm that Prmt7 activity is required to suppress ER stress in cardiac tissue and endothelial cells.

[0128] In other words, it can be seen that Prmt7 deficiency in endothelial cells exacerbates cardiac dysfunction caused by myocardial infarction, and Prmt7 inhibition exacerbates ER stress caused by cardiac cell death due to myocardial infarction.

[0129]

[0130] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

Claims

1. A pharmaceutical composition for the prevention or treatment of heart disease comprising: a Prmt7 protein; a gene encoding the Prmt7 protein or an analog thereof; or a preparation that promotes the expression or activity of the gene encoding the Prmt7 protein.

2. In Paragraph 1, A composition characterized in that the heart disease is an ischemic heart disease selected from myocardial infarction (MI), ischemic cardiomyopathy, myocardial ischemia, and angina pectoris; or fibrosis.

3. In Paragraph 1, The above composition is a composition that promotes the proliferation of endothelial cells (EC) or inhibits their death.

4. In Paragraph 1, The above composition is a composition that inhibits stress of the endoplasmic reticulum (ER).

5. A health functional food composition for the prevention or improvement of heart disease comprising: a Prmt7 protein; a gene encoding the Prmt7 protein or an analog thereof; or a preparation that promotes the expression or activity of the gene encoding the Prmt7 protein.

6. In Paragraph 5, A composition characterized in that the heart disease is an ischemic heart disease selected from myocardial infarction (MI), ischemic cardiomyopathy, myocardial ischemia, and angina pectoris; or fibrosis.

7. In Paragraph 5, The above composition is a composition that promotes the proliferation of endothelial cells (EC) or inhibits their death.

8. In Paragraph 5, The above composition is a composition that inhibits stress of the endoplasmic reticulum (ER).

9. A health functional food composition for protecting endothelial cells (EC), comprising: a Prmt7 protein; a gene encoding the Prmt7 protein or an analog thereof; or a preparation that promotes the expression or activity of the gene encoding the Prmt7 protein.

10. In Paragraph 9, The above-mentioned endothelial cell protective composition is a health functional food composition that improves vascular function. 11.1) Step of treating endothelial cells (EC) with a candidate substance; 2) a step of measuring the expression or activity of the Prmt7 protein in endothelial cells treated with the above candidate substance; and 3) A screening method for a therapeutic substance for heart disease, comprising the step of selecting a substance in which the activity or expression of the Prmt7 protein is increased compared to a control group not treated with the candidate substance as a therapeutic substance for ischemic heart disease.

12. An animal model that increases the occurrence or progression of heart disease by specifically inducing a decrease in the expression of the Prmt7 protein or the gene encoding the Prmt7 protein in cardiac cells.

13. In Paragraph 13, The above animal model is an ischemic heart disease selected from myocardial infarction (MI), ischemic cardiomyopathy, myocardial ischemia, and angina pectoris; or an animal model having increased occurrence or progression of fibrosis.

Images