KR20210070783A - Pharmaceutical composition for preventing or treating heart failure with preserved ejection fraction - Google Patents

Abstract

The present invention relates to a composition for preventing or treating heart failure, preserving ejection fraction. More specifically, the present invention contains N(ω)-propyl-L-arginine or a pharmaceutically acceptable salt thereof, thereby inhibiting NO production by nNOS and inhibiting nitrosylation of HDAC2 to exhibit an excellent medicinal effect on heart failure.

Description

PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING HEART FAILURE WITH PRESERVED EJECTION FRACTION}

The present invention relates to a composition for preventing or treating heart failure.

Heart failure is a disease that occurs when the heart fails to properly supply blood to peripheral organs due to structural or functional abnormalities of the heart. Heart failure can result from dysfunction of the heart, either systolic or diastolic.

The most well-known type of heart failure may be systolic heart failure, in which the myocardium used to contract is lost. Patients with systolic heart failure are characterized by an ejection fraction of less than 40% as a result of echocardiography. Therefore, systolic heart failure is also known as heart failure with reduced ejection fraction (HFrEF). The main cause of myocardial dysfunction in HFrEF is myocardial infarction as a result of coronary artery disease. Many HFrEF patients present with symptoms such as shortness of breath, limited exercise capacity, or coughing.

Interestingly, many heart failure patients present with similar symptoms such as shortness of breath, limited exercise capacity, or coughing, but with well-preserved ejection fractions of 50% or more. In these patients, systolic function is maintained fairly well, while diastolic function is somewhat variable. In recent decades, the number of HFpEF patients has been increasing in developed countries, and the clinical and social burden of HFpEF has increased at the same time. In particular, as 80 to 90% of HFpEF patients suffer from hypertension, hypertension is considered the most important risk factor.

Historically, HFpEF has been referred to as diastolic heart failure. However, recent findings suggest a more complex pathophysiological mechanism. The diastolic function of HFpEF patients has been widely reported, and there are even cases where it is normal. Thus, although they share many clinical features, diastolic dysfunction and HFpEF are not taken as synonymous.

Unfortunately, none of the therapies that have been proven effective for HFrEF have been demonstrated to be beneficial in increasing the survival rate of patients with HFpEF. Even nitric oxide (NO) donors, which are potent vasodilators, have no benefit in the treatment of HFpEF, which raises the fundamental question whether a strategy to supplement NO is an appropriate treatment.

Korean Patent No. 1922542

An object of the present invention is to provide a composition for preventing or treating heart failure.

An object of the present invention is to provide a health functional food for preventing or improving heart failure.

1. A pharmaceutical composition for preventing or treating heart failure, comprising N(ω)-propyl-L-arginine (N(ω)-propyl-L-arginine; NPLA) or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to the above 1, wherein the heart failure is heart failure with preserved ejection fraction.

3. A health functional food for preventing or improving heart failure, comprising N(ω)-propyl-L-arginine or a pharmaceutically acceptable salt thereof.

4. The health functional food according to the above 3, wherein the heart failure is heart failure with preserved ejection fraction.

The composition of the present invention inhibits NO production by nNOS and inhibits nitrosylation of histone deacetylase 2 (HDAC2), thereby exhibiting excellent drug efficacy and health functionality for heart failure preserving ejection fraction.

1 shows the mechanism by which HFpEF is induced.
Figure 2 shows the change in ejection fraction, E / E' ratio change and exercise capacity (latency time) in SAUNA mouse construction and SAUNA mouse as a HFpEF model.
Figure 3 shows the change in ejection fraction, E/E' ratio change, and exercise capacity (latency time) in mTAC and mild transverse aortic constriction (mTAC) mouse construction with the HFpEF model.
4 shows the results of biotin-switching assay showing the degree of S-nitrosylation in the heart of HFpEF and the expression level of nNOS in the HFpEF model.
5 shows the results of S-nitrosylation of HDAC2 by nNOS in an in vitro cell model.
6 shows the results of confirming whether the HDAC2 enzyme activity is changed according to S-nitrosylation and nitrosylation of HDAC2 in vitro.
7 shows S-nitrosylation of HDAC2 in an in vivo HFpEF model.
8 shows the results of confirming whether the HDAC2 enzyme activity changes according to S-nitrosylation and nitrosylation of HDAC2 in an in vivo HFpEF model.
9 shows the results showing that S-nitrosylation of C262 and C274 of HDAC2 aggravates HFpEF.
10 shows the results showing the resistance to mTAC of HDAC2 2CA mice.
11 shows the results showing that denitrosylation of HDAC2 induced by NRF2 alleviates HFpEF.

The present invention provides a pharmaceutical composition for preventing or treating heart failure, comprising N(ω)-propyl-L-arginine or a pharmaceutically acceptable salt thereof.

N(ω)-propyl-L-arginine may be derived from nature or synthesized using a known chemical synthesis method.

The pharmaceutically acceptable salt may be an acid addition salt formed with a free acid. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids.

Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodine. Id, fluoride, acetate propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propylate, oxalate, malonate, succinate, suberate, seba Kate, fumarate, maleate, butyne-1,4-dioate, nucleic acid-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate Eight, phthalate, terephthalate, benzene sulfonate, etuene sulfonate, chlorobenzene sulfonate, xylene sulfonate, phenyl acetate, phenyl propionate, phenyl butyrate, citrate, lactate, β-hydroxybutyrate, glycolate , may include maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt can be prepared by a conventional method, for example, by dissolving the compound in an excess of an aqueous acid solution and precipitating the salt using a hydrating organic solvent such as methanol, ethanol, acetone or acetonitrile. It can also be prepared by heating the compound and an acid or alcohol in water and then evaporating the mixture to dryness or by suction filtration of the precipitated salt.

In addition, a pharmaceutically acceptable metal salt can be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, as the metal salt, it is pharmaceutically suitable to prepare sodium, potassium or calcium salts. Also, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg silver nitrate).

The pharmaceutical composition of the present invention may be usefully used for the prevention or treatment of heart failure. Heart Failure (HF) is a disease in which the pumping function of the heart fails to properly supply blood to the body, and may occur due to dysfunction of the systolic or diastolic functions of the heart.

Heart failure can be acute or chronic, as well as from a variety of causes such as cardiovascular disease, myocardial infarction, hypertension, heart valve disease, myocardial disease (eg definitive cardiomyopathy, hypertrophic cardiomyopathy) or myocarditis, endocarditis, congenital heart disease, chronic heart failure due to lung disease, diabetes, or arrhythmia, but is not limited thereto.

Heart failure may be systolic heart failure, diastolic heart failure, congestive heart failure, heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, and the like, preferably heart failure with preserved ejection fraction, but is not limited thereto.

The term 'prevention' of the present invention means any action that inhibits or delays the invention of a related disease by administering the composition of the present invention. Those of ordinary skill in the art to which the present invention pertains will be able to determine that related diseases can be prevented by administering the composition of the present invention before or at an early stage of symptom onset.

The term 'treatment' of the present invention refers to any action that improves or beneficially changes the symptoms of a related disease by administering the composition of the present invention, and treatment includes alleviation or improvement. Those of ordinary skill in the art to which the present invention pertains will be able to know the exact criteria of the disease with reference to the data presented by the Korean Medical Association, etc., and determine the degree of improvement, improvement, and treatment.

The pharmaceutical composition of the present invention may include an active ingredient alone, or may further include one or more pharmaceutically acceptable carriers, excipients or diluents.

The term 'pharmaceutically acceptable' of the present invention means that it exhibits non-toxic properties to cells or humans exposed to the composition of the present invention.

The pharmaceutical composition of the present invention may further include a known heart failure treatment substance. That is, the active ingredient of the pharmaceutical composition of the present invention may be administered in combination with a known substance having a preventive or therapeutic effect for heart failure, particularly heart failure with preservation of ejection fraction.

Such a known material may be DNA, RNA, a compound, a protein, or a polypeptide, but is not limited thereto.

The term 'administration' of the present invention means introducing a predetermined substance to an individual by an appropriate method, and the term 'subject' of the present invention includes humans who have or may develop heart failure (eg, heart failure with preserved ejection fraction). It means any animal such as rats, mice, livestock, etc. As a specific example, it may be a mammal including a human.

The route of administration of the pharmaceutical composition of the present invention is, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or work is included.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and when administered parenterally, external skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection may be selected, but limited thereto it's not going to be

A preferred dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the degree of disease, drug form, administration route and period, but may be appropriately selected by those skilled in the art. The pharmaceutical composition of the present invention may be administered at, for example, 0.01 to 1000 mg/kg/day, for example, 0.1 to 500 mg/kg/day.

The dosage does not limit the scope of the present invention in any way. The number of administration can be administered once a day or divided into several times within a desired range, and the administration period is not particularly limited.

In the case of formulating the pharmaceutical composition of the present invention, it is prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract, such as starch, calcium carbonate, sucrose, or lactose ( lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycero geratin, and the like can be used.

The present invention provides a health functional food for preventing or improving heart failure, comprising N(ω)-propyl-L-arginine or a pharmaceutically acceptable salt thereof.

Since the contents of N(ω)-propyl-L-arginine, salt or heart failure have been described above, a detailed description thereof will be omitted.

The health functional food of the present invention may further include one or more of carriers, diluents, excipients and additives, and may be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules and liquid formulations. .

Additives include natural carbohydrates, flavoring agents, nutrients, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), coloring agents, fillers (cheese, chocolate, etc.), lactic acid and its salts, alginic acid and its salts. , organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohol, carbonation agent and at least one component selected from the group consisting of pulp can be used. Natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides, for example, conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Flavoring agents can be natural flavoring agents (taumatine, stevia extract (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).

The health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof , organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the composition of the present invention may contain the pulp for the production of natural fruit juices and vegetable beverages.

Specific examples of carriers, excipients, diluents and additives include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate Kate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate And it may be at least one selected from the group consisting of mineral oil.

The health functional food of the present invention may be formulated using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant.

Hereinafter, examples will be given to describe the present invention in detail.

Experimental method

antibody

The antibodies used were: mouse monoclonal anti-HDAC2 (Abcam, Cambridge, UK, 1:5,000, 12169), rabbit polyclonal anti-HDAC2 (Invitrogen, Carlsbad, CA, 1:1,000, 51-5100). ), mouse monoclonal GAPDH (Bio-Rad, Hercules, CA, 1:1,000, VMA00046), rabbit polyclonal anti-GAPDH (Bio-Rad, 1:1,000, VPA00187) and mouse monoclonal anti-HA ( Sigma, St. Louis, MO, 1:30,000, H9658).

virus

Adenovirus expressing nNOS and AAV9-NRF2 was purchased from a specialized manufacturing company (Vector Biolabs, PA, Malvern, PA). nNOS: ADV-216899, AAV-NRF2: AAV-216638, adenovirus NRF2 were provided by Professor Lee In-gyu, Kyungpook National University College of Medicine.

animal model

The use of animal experiments for disease models was approved by the Research Institutional Animal Care and Use Committee, Chonnam National University College of Medicine. For HFpEF model induced SAUNA (SA lty drinking water / U nilateral ephrectomy N / A ldosterone) model in an 8-week-old male C57BL / 6 mice. Mice were anesthetized with 2,2,2-tribromoethanol (300 mg·kg ^-1 , Sigma, T48402) and placed in a lateral decubitus position. The left kidney was removed and a micro-osmotic pump (Alzet®, Durect Corp, Cupertino, CA, 1004) containing d-aldosterone (Sigma, 0.30 μg h ^{−1 , A9477) was implanted subdally.} From day 1 after surgery, mice were provided with drinking water containing 1% NaCl for 30 days. Exercise capacity and cardiac function were assessed on day 30.

For the HFpEF alternative animal model, mild TAC was performed to induce isolated diastolic dysfunction without change in contractile function. A partial thoracotomy was performed on the second rib and two loose knots were made with a 26 gauge needle between the brachiocephalic artery and the left common carotid artery.

genetically engineered mouse

HDAC2 S-nitrosylation-dead knock-in mice (HDAC2 2CA) were produced by a company using CRISPR/Cas9 technology (Tulgen, Korea). The mouse HDAC2 genome sequence around cysteine 262 (C262) and cysteine 274 (C274) was replaced from TGT GGC GCA GAC TCC CTG TCT GGG GAC AGG CTT GGT TGT to GCT GGA GCC GAT AGC CTT AGC GGA GAT CGC CTG GGA GCT. Except for two targeted cysteines, the protein sequence was unchanged (CGADSLSGDRLGC to AGADSLSGDRLGA).

Genotyping was performed with T7E1 (NEB, Ipswich, MA, M0302). Direct Sanger sequencing of the PCR product was performed to further confirm the genotype of the homozygote. The following oligomer sets were used for genotyping: sense: 5'-TGCTGTCAATTTTTCCCATGA-3', antisense: 5'-AGAGTTTGGCATCGAGTTGG-3'.

In vivo drug administration and gene delivery

N(ω)-propyl-L-arginine (NPLA, Cayman, Ann Arbor, MI, 80587) (50 mg·kg ^-1 ·day ^-1 , every other day) was injected to inhibit nNOS.

AAV9-NRF2 infection (Vector Biolabs, Malvern, PA) was performed via direct cardiac injection. C57BL/6 mice were anesthetized with 2,2,2-tribromoethanol and maintained on a ventilator. The intercostal space between the fourth and fifth ribs was cut and expanded. ^{1×10 12} copies of AAV9-NRF2 were injected into the left ventricle vitreous wall. For successful expression of infected AAV9-NRF2, AAV virus was injected 2 weeks prior to SAUNA surgery.

Echocardiogram

Cardiac function was measured by ultrasonography (General Electric Company, Chicago, IL, Vivid S5). Mice were anesthetized with 2,2,2-tribromoethanol and checked for non-response with a light touch. Two-dimensional M-modes were obtained from cross-sternal long-axis or short-axis cross-sections at the level of the papillary muscle.

The ejection fraction was determined by the Teichholz equation: EF (%) = (Vd-Vs) / Vd, where Vd is end-diastolic left ventricular volume and Vs is end-systolic left ventricular volume, Vd = [7 / (2.4 + LVIDd) ] × LVIDd ³ , Vs = [7 / (2.4 + LVIDs)] × LVIDs ³ , where LVIDd is the end-diastolic LV ventricular size and LVIDs is the end-systolic ventricular size.

E and E' were measured to evaluate diastolic function. After measuring the short sternal cross-section, the ultrasound probe was tilted 45° to visualize the sternal 4-chamber cross-section. The mitral valve E wave at the mitral valve opening was recorded in pulse wave mode. Mitral valve ring motion, also known as E' wave, was assessed with tissue velocity imaging mode.

Rotarod treadmill test

The locomotor tolerance of mice was measured using a rotarod system (ENV-577M, Med Associate Inc, Fairfax, VT). Mice were pre-adapted to a rod at a fixed speed of 10 rpm for 5 min prior to measurement. Mice were exercised on a round cylinder (35 mm in diameter) that was continuously accelerated from 4 to 40 rpm at a rate of 1 rpm per 5 s. Motor ability was evaluated as the time until passive rotation while holding the rotating rod or falling from the rotating rod for up to 5 minutes. After three repeated measurements, the longest exercise time was used in the study. The time interval between each measurement was 30 minutes.

cell culture

H9c2 (CRL-1446) and 293T (CRL-3216) were obtained from the American Type Culture Collection (ATCC, Manassas, Va.). For primary cultured cells, neonatal rats were used. Hearts were harvested from 1–2 day old Sprague Dawley rats, and only the ventricles were collected after the aorta and both atria were removed. The ventricles were washed in ADS buffer (20 mM HEPES pH 7.4, 120 mM NaCl, 5.5 mM glucose, 11 mM NaH ₂ PO ₄ , 5.4 mM KCl in distilled water) containing 0.1% type 2 collagenase (Worthington, Lakewood, NJ, LS004177). and 0.44 mM MgSO ₄ ) at 37° C. for 30 min. The enzymatic reaction was terminated by addition of normal growth medium. Cells were precipitated with 400 RCF for 5 min. The collected cells were resuspended in normal growth medium and pre-incubated for 1 h to remove fibroblasts. Neonatal rat ventricular cardiomyocytes were counted and plated for study.

Adult Cardiomyocyte Isolation

Adult mouse ventricular cardiomyocytes were obtained from C57BL/6 mouse hearts. Mice were injected with 50 units of heparin and euthanized by cervical dislocation. Hearts were rapidly harvested and calcium-free thyroid buffer (10 mM HEPES pH 7.4, 137 mM NaCl, 5.4 mM KCl, 1 mM MgCl ₂ , 10 mM glucose, 5 mM taurine and 10 mM 2,3-butanedione monooxime) was perfused. Enzymatic digestion with digestion buffer (calcium-free tyroid buffer supplemented with hyaluronidase [Worthington, 0.1 mg.ml- ¹ , LS005477] and collagenase type B [Sigma, 0.35 U.ml- ^{1, 11088807001])} was performed. After 10 minutes of digestion, the left ventricle was collected and cut into small pieces. Further digestion was performed with gentle stirring for 10 minutes. The cells were briefly allowed to settle for large fractions and the supernatant was filtered using a 100-mm pre-cell filtration net. The filtered cells were plated on a culture dish for 2 hours to remove cardiac fibroblasts.

Immunoprecipitation

For immunoprecipitation, 1 mg of 1% NP buffer (1% Igepal CA-630, 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, protease inhibitor mixture (Gendepot, Barker, TX, P3100))). Cell lysates were prepared. 1 μg of primary antibody was added and reacted for 2 hours with continuous rotation at 4°C.

Antibodies were collected by adding 30 μL of Protein G Plus beads (Santa Cruz Biotechnology, Santa Cruz, CA, SC-2002). After 2 hours of continuous rotation at 4°C, the beads were precipitated by light centrifugation and washed twice with 1% NP buffer. NuPAGE SDS sample buffer (Invitrogen, NP0007) was added with beta-mercaptoethanol (Sigma, 63689), and boiled for 5 minutes to denaturate and reduce the precipitate. Proteins were separated by SDS-PAGE and 30 μg of cell lysate was used as a control for immunoprecipitation.

Biotin-substitution assay

Protein S-nitrosylation was determined by biotin substitution technique. All reactions were performed in dark amber tubes to avoid UV exposure. Protein lysates were prepared with HENS buffer (250 mM HEPES, pH 7.7, 1 mM EDTA, 0.1 mM neocuproin [Sigma, P9599], 1% SDS) containing a protease inhibitor cocktail. Cell lysates or cardiac lysates were prepared with 200 μL of HENS buffer and contained 100 μg or 500 μg of protein, respectively. Block free thiol groups with a blocking solution (250 mM HEPES, pH 7.7, 1 mM EDTA, 0.1 mM neocuproin, 1% SDS and 25 mM S-methylmethanethiosulfonate [Sigma, 208795]) for 1 h at room temperature. did it The blocking step was terminated by adding 1 mL of ice-cold acetone. Proteins were precipitated by centrifugation at 6,000 RCF for 15 min. After removal of acetone, the precipitate was dissolved in 170 μL of HENS buffer. After adding 10 μL of 1 M sodium ascorbate, it was left for 20 minutes to remove S-NO, and then 20 μL of 4 mM biotin-HPDP (Thermo, MA, Waltham, MA, 21341) was added to label biotin 40 min. The reaction was stopped by ice-cold acetone precipitation. Biotin-labeled protein was dissolved in 400 µL of HENS buffer and neutralized with 800 µL of 1% NP buffer containing protease inhibitor cocktail. Biotin-labeled protein was collected by adding 30 μL of streptavidin-agarose beads and continuous rotation at 4° C. overnight. Beads were washed 3 times with 1% NP buffer and samples were analyzed by SDS-PAGE.

mass spectrometry

Liquid chromatography mass spectrometry was used to screen for nitrosylated substrates of nNOS in the heart. After biotin-labeled protein was collected with streptavidin-agarose beads, it was separated on a 4-12% Bis-Tris gel (Bolt™, Invitrogen, NW0412BOX), and the protein was visualized by Silver staining method (Pierce™, Thermo, 24612). did. In the conditions of Ad-nNOS infection or 500 μM GSNO (Cayman, 82240) treatment conditions, darkened or newly appeared bands were cleaved, and mass spectrometry was requested. Nano LC-MS/MS analysis was performed at the Korea Basic Science Institute (Ochang Biomedical Omics Research Center, Korea). 14-3-3, Actin, Atp1a1, Fabp5, Gapdh, HDAC2, Hsp90α/βNme1/2, Nos1, Pdia3, Pdlim5, Phgdh, Ppia, Serpinh1, Sod1, Stip1, Tcp1, Tubulin and Uba3 were identified.

Histone deacetylase activity assay

HDAC2 activity was measured using a commercial kit (HDAC-Glo ^™ 2 Assays, Promega, Madison, WI, G9590). Cardiac lysates were prepared with 1% NP buffer without EDTA to avoid zinc chelation. 50 μg of cardiac lysate was mixed with HDAC2 assay substrate and incubated for 15 min at room temperature. To confirm the S-nitrosylation effect of HDAC2, 500 μM of GSNO or 500 μM of GSH (L-Glutathione, Cayman, 10007461) was added to the lysate and incubated at room temperature for 30 minutes. As a negative control, 500 nM of tricostatin A (Sigma, T8552), a potent HDAC inhibitor, was added to the assay buffer. The deacetylase activity of HDAC2 was measured with a luminometer. The solvent-treated state was considered 1 and the multiplier was calculated.

Quantitative real-time polymerase chain reaction

Total mRNA was extracted with TRIzol (Invitrogen, 15596026). cDNA was synthesized using a random hexamer (M-MLV reverse transcriptase, Invitrogen, 28025013). Quantitative real-time PCR was performed using QuantiTect SYBR Green kits (Qiagen, Hilden, Germany, 204143) with Rotor-Gene Q (Qiagen). PCR analysis was performed in triplicate and the average was considered as a single result. The relative expression level of specific mRNA was calculated by correcting the expression level of GAPDH. Specific oligomer sets are shown in Table 1 below.

division set order mouse GAPDH sense 5'-GCATGGCCTTCCGTGTTCCT-3' antisense 5'-CCCTGTTGCTGTAGCCGTAT-3' mouse nNOS sense 5'-ACTGACACCCTGCACCTGAAGA-3' antisense 5'-GTGCGGACATCTTCTGACTTCC-3' mouse iNOS sense 5'-CAGCTGGGCTGTACAAACCTT-3' antisense 5'-CATTGGAAGTGAAGCGGTTCG-3' mouse eNOS sense 5'-CCTCGAGTAAAGAACTGGGAAGTG-3' antisense 5'-AACTTCCTTGGAAACACCAGGG-3' Mouse GSTA3 sense 5'-TACTTTGATGGCAGGGGAAG-3' antisense 5'-GCACTTGCTGGAACATCAGA-3' mouse 18s sense 5'-GTAACCCGTTGAACCCCATT-3' antisense 5'-CCATCCAATCGGTAGTAGCG-3' Human NRF2 sense 5'-CACATCCAGTCAGAAACCAGTGG-3' antisense 5'-GGAATGTCTGCGCCAAAAGCTG-3' Rat NRF2 sense 5'-AGAAGCACACTGAAGGCACGG-3' antisense 5'-GAATGTTGTTGGCTGTGCTTTAGGTC-3' Rat GSTA3 sense 5'-AGTCCTTCACTACTTCGATGGCAG-3' antisense 5'-CACTTGCTGGAACATCAAACTCC-3' Rat GAPDH sense 5'-ATGACATCAAGAAAGGTGGTG-3' antisense 5'-CATACCAGGAAATGAGCTTG-3'

statistics

Statistical significance was analyzed with PASW Statistics 25 (SPSS, IBM corp, IL, Chicago). For two independent groups, a two-tailed unpaired Student's t-test was applied after confirming the normal distribution. For groups of three or more, one-way analysis of variance was used. When equality of variance was confirmed using Levene's statistics, Tukey's post-hoc test was applied in multiple comparisons, and Dunnett T3 was used as a post-hoc test when equal variances could not be applied. Survival rates were visualized and calculated using Prism 8.0 (GraphPad, San Diego, CA). Significance was determined at p <0.05.

Experiment result

Establishment of the HFpEF animal model: SAUNA and mTAC

To investigate the pathophysiological mechanism of HFpEF, we first constructed the SAUNA model, known as one of the animal models most similar to human HFpEF (see Figure 2A). On the 30th day after the initiation of the protocol, cardiac function was measured by checking the ejection fraction (E) and mitral valve ring movement (E') in the early diastole and the ability to perform the rotarod treadmill test. When looking at ejection fraction, contractile function was well preserved in the SAUNA group (see Figure 2B). However, E/E' was abnormally increased (see Figure 2C). Also, motor capacity was reduced in SAUNA mice, suggesting exercise intolerance (see Figure 2D). mTAC was introduced as an alternative model and cardiac function was evaluated 4 weeks later. In the case of mTAC, HFpEF was induced within 1 month, and the ejection fraction did not change, but E/E' and exercise capacity were changed (see FIG. 3). Through this, it was confirmed that the SAUNA and mTAC models for the study of HFpEF were successfully established.

Confirmation of increased S-nitrosylation in HFpEF cardiomyocytes

Post-translational modulation of proteins was investigated in both mouse models. We first evaluated acetylation, methylation and phosphorylation using an immunoprecipitation-based assay. Acetylation or methylation did not fluctuate significantly. Although the general phosphorylation state appeared to be increased, the overall change was not dramatic. On the other hand, as shown in the biotin-substitution analysis result, it was confirmed that protein S-nitrosylation was significantly increased in the HFpEF heart (see FIG. 4A ).

NO can be produced by three types of nitric oxide synthase: nNOS (NOS1), iNOS2 (NOS2), and eNOS (NOS3).

To confirm the source of NO, the amount of transcription of nNOS, iNOS, and eNOS was measured by quantitative real-time PCR. In mTAC mice, both nNOS and iNOS increased rapidly (see Fig. 4B), whereas only the transcription amount of nNOS increased in the SAUNA model (see Fig. 4C). In particular, it was confirmed that nNOS was predominantly increased in cardiomyocytes isolated from SAUNA-treated mice among cardiac cells (see Fig. 4D).

Improvement of HFpEF diastolic dysfunction by nNOS inhibition

N(ω)-propyl-L-arginine was administered to the HFpEF model to determine whether nNOS is involved in HFpEF. N(ω)-propyl-L-arginine was administered every other day as described in 'In vivo drug administration and gene transfer'.

Figure 4E shows echocardiography of mitral valve E and E' waves, reflecting ventricular spasticity and diastolic dysfunction. SAUNA induced an increase in E/E' (see Figure 4F) and a decrease in exercise tolerance as measured by the exercise capacity of the rotarod test (see Figure 4G), which were normalized by treatment with NPLA. These results indicate that nNOS, the target of NPLA, is involved in diastolic dysfunction and impairment of motor ability in HFpEF, and that NPLA can ameliorate the impairment.

Target identification of nNOS-mediated nitrosylation

A biotin substitution assay was used to identify nNOS specific S-nitrosylation targets in the heart.

H9c2 cells were infected with adenovirus GFP (Ad-GFP) or adenovirus nNOS (Ad-nNOS), and S-nitrosylated cysteine (SNO-cysteine) was labeled with biotin. After sedimentation with 30 μL of streptavidin-agarose beads, biotinyl protein was separated on an SDS-PAGE gel and visualized by silver staining. Darkened or newly appeared bands in Ad-nNOS were sequenced by mass spectrometry. As shown in Fig. 5A, HDAC2 (upper arrow) and GAPDH (down arrow) were identified. That is, it was confirmed that S-nitrosylation occurred when H9c2 cells were infected with Ad-nNOS (see Fig. 5A).

Similarly, incubation of cardiac lysates with S-nitrosoglutathione (GSNO), a non-selective NO donor, increased S-nitrosylation (see Figure 5B).

Proteins with increased nitrosylation were identified in lysates obtained from Ad-nNOS infected cardiomyocytes (see Fig. 5A) and hearts (see Fig. 5B) by mass spectrometry. Western blot confirmed that HDAC2 was the target of S-nitrosylation; Biotin substitution analysis showed that HDAC2 was S-nitrosylated in the presence of sodium ascorbate and biotin (see 4th lane in Figure 5C). Treatment with GSNO increased S-nitrosylation of HDAC2 (see 3rd lane in Figure 6A). Infection with Ad-nNOS increased S-nitrosylation of HDAC2 (see Figure 5D) and GAPDH (see Figure 5E). On the other hand, GSNO (see Figure 6B) treatment or Ad-nNOS infection (see Figure 6C) did not decrease the enzymatic activity of HDAC2.

HDAC2 S-nitrosylation in the HFpEF model heart

Based on the induction of nNOS into cardiomyocytes (see Figure 4C and Figure 4D), S-nitrosylation was increased in HFpEF hearts (see Figure 4A), and infection with adenovirus nNOS (Ad-nNOS) resulted in S-nitrosylation of HDAC2. Thus (see Figure 5D), it was hypothesized that HDAC2 was S-nitrosylated in the HFpEF heart.

Biotin-displacement analysis using SAUNA cardiac lysates showed a significant increase in S-nitrosylation of HDAC2 (see Figures 7A and 7B). To determine whether the increase in S-nitrosylation of HDAC2 is dependent on nNOS activity, NPLA was administered to HFpEF mice. It was confirmed that S-nitrosylation of HDAC2 was almost completely eliminated in SAUNA mice by administration of NPLA (see Fig. 7C). S-nitrosylation of HDAC2 was also observed in the mTAC model (see Figure 8A). However, HFpEF stress did not affect HDAC2 enzymatic activity in SAUNA mouse hearts (see Figure 8B).

Confirmation of NO transport from the cytoplasm of cardiomyocytes to nuclear HDAC2

nNOS is mainly localized to the cytoplasmic membrane, whereas HDAC2 is present in the nucleus. Thus, the question of how membrane-bound nNOS can S-nitrosylate nuclear HDAC2 may be raised. It was hypothesized that GAPDH could indirectly S-nitrosylate HDAC2 by transferring NO from membrane nNOS to nuclear HDAC2. We first investigated whether nNOS-mediated S-nitrosylation of GAPDH induces redistribution in cardiomyocytes; Infection with Ad-nNOS significantly increased the intranuclear accumulation of GAPDH (see Figure 7D).

Immunoprecipitation assay was performed to confirm whether S-nitrosylation of GAPDH induces physical interaction with HDAC2 for NO transport. Infection with Ad-nNOS increased the physical interaction between GAPDH and HDAC2 (see Figure 7E). Interestingly, the physical interaction between GAPDH and HDAC2 was also enhanced when GSNO, a non-selective nitrosylation-inducing agent, was incubated with cardiac lysates (see Figure 7F).

S-Nitrosylation of HDAC2 C262/C274

To understand the role of HDAC2 S-nitrosylation in HFpEF, we identified residues capable of nNOS-mediated S-nitrosylation and investigated whether inhibition of S-nitrosylation affects HFpEF induction. HDAC2 has four cysteine residues for S-nitrosylation: C101, C262, C274 and C285. Cysteine residues that can be S-nitrosylated may vary depending on the cell type. For example, in HeLa cells all four residues are S-nitrosylated, whereas in neurons C262 and C274 are S-nitrosylated. Of note, SNO-induced overall S-nitrosylation of HDAC2 in skeletal muscle leads to a decrease in enzymatic activity, whereas S-nitrosylation of C262 and C274 does not induce a decrease in enzymatic activity. These results raise the possibility that C101 and/or C285 are involved in enzymatic activity and C262 and C274 are not.

As shown in FIG. 6 , GSNO did not reduce HDAC2 activity, but sufficiently induced S-nitrosylation of HDAC2. In addition, nNOS was significantly induced in SAUNA and mTAC mouse hearts (see Figures 4A and 4C). Considering the previous report that nNOS induces S-nitrosylation of HDAC2 C262/C274 in neurons, HDAC2 C262 and C274 are likely targets of S-nitrosylation. Therefore, experiments were conducted focusing on C262 and C274.

The C262 and C274 residues were highly conserved across various species (see Figure 9A). S-nitrosylation resistant mutant mice were produced by replacing C262 and C274 with alanine by gene-knock-in technology (hereinafter, HDAC2 2CA). S-nitrosylation of HDAC2 could not be observed in HDAC2 2CA mouse embryonic fibroblasts in the presence of adenovirus nNOS-HA (see Fig. 9B). This suggests that C262 and C274 are the only cysteines responsible for S-nitrosylation of HDAC2. Then, the SAUNA model was applied to HDAC2 2CA mice. In contrast to wild-type (see Figure 7A), SAUNA failed to induce S-nitrosylation of HDAC2 in HDAC2 2CA hearts (compare lanes 2-5 with lanes 6-10 of Figure 9C). Not even a basal level of S-nitrosylation was observed when compared to the control (compare lanes 2-5 and 1 in FIG. 9C).

Next, it was confirmed whether S-nitrosylation inhibition of 2CA HDAC2 affects HFpEF. Resistance to SAUNA stress was observed in HDAC2 2CA mice. The abnormal increase in E/E' observed in wild-type mice was not observed in HDAC2 2CA mice (see Figure 9D). HDAC2 2CA mice also did not develop SAUNA-induced impairment of exercise tolerance (see Figure 9E). Even under SAUNA stress, contractile function was well preserved in HDAC2 2CA mice (see FIG. 9F ). mTAC also failed to induce diastolic dysfunction in HDAC2 2CA mice (see Figure 10A). Through these results, it was confirmed that HDAC2 2CA mice were resistant to HFpEF.

In addition, by measuring the HDAC2 activity, it was confirmed whether S-nitrosylation affects the HDAC2 function of the mouse heart. The HDAC inhibitor tricostatin A (TSA) successfully inhibited the enzymatic activity of HDAC2 (see second bar in Figure 10B). However, the deacetylation activity of HDAC2 in cardiac lysates was not altered by GSNO-induced nitrosylation (see 4th bar in Figure 10B). These results suggest that S-nitrosylation of HDAC2 does not directly affect the activity of HDAC2.

HFpEF mitigation through NRF2-induced denitrosylation of HDAC2

Through the above-described experimental results, the role of nNOS related to HDAC2 C262/C274 S-nitrosylation in HFpEF was confirmed. In addition, it was confirmed that nNOS inhibition can block HFpEF in vivo. In addition, additional experiments were conducted to confirm whether deS-nitrosylation of HDAC2 could alleviate HFpEF.

NRF2 is known to act as an antioxidant and induce protein denitrosylation. First, it was confirmed whether NRF2 affects the nitrosylation of HDAC2 in cardiac cells, and it was confirmed that NRF2 can denitrosylate HDAC2. Infection with adenovirus NRF2 (see Fig. 11A) significantly activated its target gene, glutathione S-transferase alpha 3 (GSTA3), in H9c2 cells (see Fig. 11B). Adenoviral nNOS-HA infection in H9c2 cells successfully induced HDAC2 S-nitrosylation, which was blocked by infection with adenovirus NRF2 (see Figure 11C).

To test the effect of NRF2-induced denitrosylation in HFpEF, the AAV9-NRF2 delivery scheme was designed (see Figure 11D). Direct cardiac infusion of AAV9-NRF2 in SAUNA mice improved diastolic function (see Figure 11E) without significant changes in systolic function (see Figure 11F). In addition, cardiac overexpression of NRF2 improved exercise tolerance (see Fig. 11G). AAV9-NRF2 infection was confirmed by an increase in the amount of transcription of the target gene GSTA3 (see Fig. 11H). As expected, S-nitrosylation of HDAC2 was significantly attenuated in the AAV9-NRF2 injected group (see Figure 11I).

Claims (4)

A pharmaceutical composition for preventing or treating heart failure, comprising N(ω)-propyl-L-arginine or a pharmaceutically acceptable salt thereof.
The pharmaceutical composition according to claim 1, wherein the heart failure is heart failure with an ejection fraction preserved.
A health functional food for preventing or improving heart failure, comprising N(ω)-propyl-L-arginine or a pharmaceutically acceptable salt thereof.
The health functional food according to claim 3, wherein the heart failure is heart failure with preserved ejection fraction.

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