GDF3 as a biomarker and biotarget in cardiac remodeling after ischemia

Abstract

Markers for the strong scarring process in the early stages after myocardial infarction (MI) have yet to be established, and identifying patients at high risk of developing most harmful fibrous remodeling and heart failure remains challenging. [Solution] Resident PW1 in scarred cardiac tissue after MI + We will demonstrate the regulation of cellular paracrine behavior and the differential abundance of 12 candidate markers in their secretomes. Of these, growth differentiation factor 3 (GDF3), a member of the transforming growth factor-β family, upregulates the proliferation of cardiac fibroblasts, which are the means of fibrosis.

Description

[Technical Field]

[0001] This invention belongs to the field of medicine, particularly cardiology. [Background technology]

[0002] Acute myocardial infarction (MI) is characterized by the death of one billion cardiomyocytes, or cardiac fibrosis, which activates the adaptive repair process, ultimately leading to the replacement of the dead cardiomyocytes with collagen-based scar tissue. 1 This leads to the regression of myocardial scarring, a key clinical predictor of mortality and sudden cardiac death. 2 Necrosis (increase in cardiac troponin) 3 and cardiac dysfunction (increased brain natriuretic peptide) 4 While markers indicating this process have been identified, markers for the robust scarring process in the early stages after myocardial infarction have yet to be identified, and identifying patients at high risk of developing most harmful fibrous remodeling and heart failure remains challenging.

[0003] Growth differentiation factor 3 (GDF3), a member of the TGF-β superfamily also known as Vgr-2, was first identified to regulate early embryonic development, adipose tissue homeostasis and energy balance by interacting with activin receptor-like kinase type I receptors B (ACVR1B, ALK4) and ACVR1C (ALK7) on the cell membrane (Andersson, O.; Korach-Andre, M.; Reissmann, E.; Ibanez, C.F.; Bertolino, P. Growth differenti-ation factor 3 signals through ALK7 and regulates accumulation of adipose tissue and diet-induced obesity. Proc. Natl. Acad. Sci. USA 2008, 105, 7252-7256). Recently, acute administration of rGDF3 to endotoxemic shock mice was shown to increase survival outcome and improve cardiac function through an anti-inflammatory response by suppressing the M1 macrophage phenotype (Wang L, Li Y, Wang X, et al. GDF3 Protects Mice against Sepsis-Induced Cardiac Dysfunction and Mortality by Suppression of Macrophage Pro-InflamMatory Phenotype. Cells. 2020;9(1):120. Published 2020 Jan 3). However, the role of GDF3 in cardiac remodeling after ischemia has never been investigated. SUMMARY OF THE INVENTION

[0004] The present invention is defined by the claims. Specifically, the present invention relates to the use of GDF3 as a biomarker and biotarget in cardiac remodeling after ischemia.

[0005] DETAILED DESCRIPTION OF THE INVENTION Regression of myocardial scar after acute myocardial infarction (MI) is a major cardiovascular research goal, but incomplete understanding of the various sources of cardiac fibrosis has been a major challenge. This incomplete understanding has, by itself, limited the identification of patient subgroups with a higher risk of developing harmful fibrotic remodeling and heart failure. Here, the inventors demonstrate the regulation of the paracrine behavior of resident PW1 + cells in scarred heart tissue after MI and the different abundances of 12 candidate markers in their secretomes. Among these, growth differentiation factor 3 (GDF3), a member of the transforming growth factor-β family, upregulates the proliferation of cardiac fibroblasts, which are a means of fibrosis. GDF3 is upregulated in scar tissue and plasma of mice and humans after myocardial infarction, and at its highest plasma concentration levels, higher fibrotic cardiac remodeling and cardiac dilation are predicted. Thus, the inventors clarify the previously uncharacterized function of GDF3 in predicting harmful fibrotic cardiac remodeling after MI.

[0006] Diagnostic method: A first object of the present invention is a method comprising determining whether a patient who has experienced a myocardial infarction has or is at risk of having harmful post-ischemic cardiac remodeling and determining the level of GDF3 in a sample obtained from the patient, wherein the level indicates whether the subject has or is at risk of having harmful post-ischemic cardiac remodeling.

[0007] As used herein, the terms "subject", "individual" or "patient" are used interchangeably and refer to any subject, particularly a human, for whom diagnosis, treatment or therapy is desired. Other subjects may include cows, dogs, cats, guinea pigs, rabbits, rats, mice, horses, etc. In some preferred embodiments, the subject is a human.

[0008] As used herein, the term “myocardial infarction” has a general meaning in the art and relates to the irreversible necrosis of the myocardium as a result of prolonged ischemia due to coronary thrombosis, i.e., the formation of a thrombus in the major blood vessels that serve the heart.

[0009] As used herein, the term “adverse post-ischemic cardiac remodeling” has a general meaning in the art and refers to significant changes that occur after myocardial infarction and may be detrimental to cardiac function. Cardiac remodeling includes molecular, cellular, and interstitial changes that clinically manifest as changes in the size, shape, and function of the heart after myocardial infarction. For example, ventricular remodeling includes progressive ventricular hypertrophy with decreased ventricular function. Myocardial function in myocardium located away from the initial infarcted myocardium is reduced. Specifically, adverse post-ischemic cardiac remodeling includes arrhythmias, diastole (assessed by left ventricular end-diastolic volume indexed by body surface area or LVEDVi), and cardiac dysfunction (left ventricular ejection fraction or EF). Typically, adverse post-ischemic cardiac remodeling is defined as an increase of more than 20% in left ventricular end-diastolic volume (LVEDV) at 6 months compared to the initial assessment (see Examples).

[0010] In some embodiments, the method of the present invention is specifically suitable for determining whether a patient is at risk of having heart failure after a myocardial infarction.

[0011] As used herein, the terms “heart failure” or “HF” have a general meaning in the art and encompass congestive heart failure and / or chronic heart failure. The functional classification of heart failure is generally performed according to the New York Heart Association Functional Classification (Criteria Committee, New York Heart Association. Diseases of the heart and blood vessels. Nomenclature and criteria for diagnosis, 6th ed. Boston: Little, Brown and co, 1964;114). In this classification, the severity of heart failure is divided into four classes (I-IV). The classes (I-IV) are as follows: Class I: No limitations in any activity; no symptoms with normal activity; Class II: Mild activity limitations; the patient is comfortable at rest or light exertion; Class III: Significant activity limitations; the patient is comfortable only at rest; Class IV: Any physical activity causes discomfort, and symptoms occur at rest.

[0012] As used herein, the term “risk” in the context of the present invention relates to the probability that an event will occur over a particular period of time, and can mean either “absolute” risk or “relative” risk of a subject. Absolute risk can be measured by referring to actual post-observational measurements for a relevant time cohort, or by referring to index values ​​developed from a statistically valid historical cohort tracked over a relevant period. Relative risk refers to the ratio of the subject’s absolute risk to either the absolute risk of a low-risk population or the mean population risk, and may vary depending on the method used to assess the clinical risk factor. The odds ratio, which is the ratio of positive to negative events for a given test outcome (odds are given by the formula p / (lp) (where p is the probability of the event occurring and (1-p) is the probability that the event will not occur)), is also commonly used for non-conversion rates. In the context of the present invention, “risk assessment” or “risk evaluation” encompasses predicting the probability, odds, or likelihood that an event or condition may occur, the likelihood that an event or condition may occur, or the incidence rate of an event or the rate of conversion from one condition to another. Risk assessment may also include predictions of future clinical parameters, values ​​of conventional experimental risk factors, or other indicators of recurrence, either absolute or relative, based on a previously measured population. The methods of the present invention may be used to perform continuous or categorical measurements of conversion risk, i.e., to diagnose and define categorical risk areas of subjects defined as being at risk of conversion. In categorical scenarios, the present invention can be used to distinguish between a normal population of subjects and other populations of subjects that are at higher risk. In some embodiments, the present invention may be used to distinguish between a population of subjects at risk from a normal population.

[0013] As used herein, the term “sample” refers to a biological sample obtained for the purpose of in vitro evaluation. A typical biological sample used in the method according to the present invention is a blood sample (e.g., a whole blood sample or a serum sample).

[0014] As used herein, the term “blood sample” means any blood sample derived from a subject. The collection of blood samples can be carried out by methods well known to those skilled in the art. In some embodiments, the blood sample is a serum sample or a plasma sample.

[0015] In some embodiments, GDF3 levels are measured 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after myocardial infarction.

[0016] As used herein, the term “GDF3” has its general meaning in the art and refers to growth / differentiation factor 3. An exemplary amino acid sequence of GDF3 is shown as SEQ ID NO: 1. Typically, the term “GDF3” is understood by the presence of a maturation domain ranging from amino acid residues at position 251 to position 364 of SEQ ID NO: 1. [ka]

[0017] The level of GDF3 in a sample can be measured using quantitative immunoassay methods known in the art, such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis.

[0018] In some embodiments, the method comprises contacting a sample with an agent that selectively binds to a mature domain (such as an antibody or an antigen-binding portion thereof) as defined above, to evaluate the protein levels in the sample. In some embodiments, the antibody has a detectable label. The antibody can be polyclonal or, more preferably, monoclonal. Intact antibodies or antigen-binding fragments thereof (e.g., Fab or F(ab’)2) can be used. As used herein, the term “labeled” with respect to an antibody encompasses directly labeling the antibody by binding a detectable substance to the antibody (i.e., physically binding), as well as indirectly labeling the antibody by reactivity with a detectable substance. Examples of detectable substances are known in the art and include chemiluminescent, fluorescent, radioactive, or colorimetric labels. For example, detectable substances can include various enzymes, complement molecule families, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable complement molecule family complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent substance includes luminol; examples of bioluminescent substances include luciferase, luciferin, aequorin, and examples of suitable radioactive substances include 125 I, 131 I, 35 S or 3 H are included.

[0019] In some embodiments, high-throughput methods, such as protein or gene chips known in the art (see, e.g., Ch. 12, “Genomics,” in Griffiths et al., Eds. Modern genetic Analysis, 1999, WH Freeman and Company; Ekins and Chu, Trends in Biotechnology, 1999; 17:217-218; MacBeath and Schreiber, Science 2000, 289(5485):1760-1763; Simpson, Proteins and Proteomics: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 2002; Hardiman, Microarrays Methods and Applications: Nuts & Bolts, DNA Press, 2003) can be used to detect the presence and / or levels of GDF3.

[0020] In some embodiments, microfluidic (e.g., "lab-on-a-chip," "micro-a-fluidic chips") devices can be used in this method for the detection and quantification of GDF3 protein in a sample. Such devices have been successfully used in microfluidic flow cytometry, continuous size-based separation, and chromatographic separation. Specifically, such devices can be used to isolate specific biological particles, such as specific proteins (e.g., GDF3), from complex mixtures such as serum (e.g., whole blood, serum, plasma). Various techniques may be used to separate GDF3 protein from heterogeneous samples. For example, some techniques allow GDF3 to be captured using a functionalized substance with a functionalized surface that binds to a target cell population. The functionalized substance may include surface-bound capture moieties such as antibodies, or other specific binding molecules such as aptamers, as is known in the art. Thus, such microfluidic chip technology may be used in diagnostic and prognostic devices for use in the methods described herein. See, for example, Lion et al., Electrophoresis 24 21 3533-3562 (2003); Fortier et al., Anal. Chem., 77(6):1631-1640 (2005); U.S. Patent Publication No. 2009 / 0082552; and U.S. Patent No. 7,611,834. This application also includes a microfluidic device comprising a GDF3 binding moiety, for example, an anti-GDF3 antibody or its antigen-binding fragment.

[0021] Typically, high levels of GDF3 indicate a higher probability that a patient will have or be at risk of having adverse post-ischemic cardiac remodeling, while low levels of GDF3 indicate a lower probability that a patient will have or be at risk of having adverse post-ischemic cardiac remodeling.

[0022] As used herein, the term “high” means a quantity that is greater than normal, greater than a given reference value or subgroup quantity, or relatively greater than a quantity in another subgroup. For example, high GDF3 refers to a quantity of GDF3 that is greater than the normal quantity of GDF3. The normal quantity of GDF3 may be determined according to any method available to those skilled in the art. High GDF3 may also refer to a quantity that is the same as or greater than a given reference value, such as a given cutoff. High GDF3 may also refer to a quantity of GDF3 in which the high GDF3 subgroup has a relatively higher level of GDF3 than another subgroup. For example, according to this specification, two different patient subgroups can be created by dividing a sample around a mathematically determined point (e.g., the median), and thus one subgroup with a higher quantity (i.e., higher than its median) and another subgroup with a lower quantity. In some cases, the "high" level may include both a very high level and a "moderately high" level, where moderately high is higher than average but lower than "very high."

[0023] As used herein, the term “low” means less than normal, less than a given reference value or subgroup quantity, or less relatively than another subgroup quantity. For example, low GDF3 means a quantity of GDF3 that is less than the normal GDF3 quantity in a particular set of patient samples. The normal GDF3 quantity may be determined according to any method available to those skilled in the art. Low GDF3 may also mean a quantity that is less than a given reference value, such as a given cutoff. Low GDF3 may also mean a quantity in a low GDF3 subgroup that is less relatively than another subgroup. For example, according to this specification, two different patient subgroups can be created by dividing a sample around a mathematically determined point (e.g., the median), and thus a group with a lower quantity (i.e., less than its median) can be created compared to another group with a higher quantity (i.e., more than its median).

[0024] In some embodiments, the method of the present invention includes the steps of: i) quantifying the level of GDF3 in a sample obtained from a patient; ii) comparing the level quantified in step i) with a predetermined reference value; and iii) concluding that if the level quantified in step i) is higher than the predetermined reference value, the patient has or is at risk of having adverse post-ischemic cardiac remodeling, or conversely, concluding that if the content quantified in step i) is lower than the predetermined reference value, the patient does not have or is not at risk of having adverse post-ischemic cardiac remodeling.

[0025] In some embodiments, the specified reference value is a threshold or cutoff value that can be determined experimentally, empirically, or theoretically. The threshold can also be arbitrarily selected based on existing experimental and / or clinical conditions, as will be recognized by those skilled in the art. For example, the specified reference value can be established using retrospective measurements in properly preserved, historically recorded subject samples. The threshold must be determined to obtain optimal sensitivity and specificity with a balance of function and benefit / risk (clinical outcomes of false positives and false negatives) of the test. Typically, the optimal sensitivity and specificity (such as the threshold) can be determined using a receiver operator characteristic (ROC) curve based on experimental data. For example, after quantifying the level of GDF3 in a sample, a classification criterion with significance for sample classification can be obtained by statistically processing the level of GDF3 measured in the sample being tested using algorithmic analysis. The formal name for the ROC curve is receiver operator characteristic curve, also known as the receiver operator characteristic curve. The receiver operator characteristic curve is primarily used in clinical biochemical diagnostic tests. The ROC curve is a comprehensive index that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specific value). The ROC curve reveals the relationship between sensitivity and specific value using image synthesis. A series of various cutoff values ​​(thresholds or critical values, boundary values ​​between normal and abnormal results in a diagnostic test) are set as continuous variables, and a series of sensitivity and specific values ​​are calculated. Next, a curve is plotted using sensitivity as the y-coordinate and specific value as the x-coordinate. The larger the area under the curve (AUC), the higher the diagnostic accuracy. In the ROC curve, the point closest to the upper left corner of the coordinate diagram is the critical point that has both high sensitivity and high specific value values. The AUC value of the ROC curve is between 1.0 and 0.5. When the AUC is greater than 0.5, the diagnostic results improve as the AUC approaches 1. When the AUC is between 0.5 and 0.7, the accuracy is low. When the AUC is between 0.7 and 0.9, the accuracy is moderate. An AUC higher than 0.9 indicates high accuracy. This algorithm is preferably performed using a computer.Existing software or systems in the art, such as MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, and GB STAT VI0.0 (Dynamic Microsystems, Inc. Silver Spring, Md., USA), may be used to plot ROC curves.

[0026] The method of the present invention is particularly suitable for identifying patients who may require additional attention and support after myocardial infarction. Specifically, the method of the present invention is suitable for determining whether a patient is eligible for treatment with vasodilators, angiotensin II receptor antagonists, angiotensin-converting enzyme inhibitors, aldosterone antagonists, diuretics, hydralazine / nitrate, antithrombolytic agents, β-adrenergic receptor antagonists, α-adrenergic receptor antagonists, calcium channel blockers, etc. Examples of ACE inhibitors include, but are not limited to, captopril, benazepril, enalapril, lisinopril, fosinopril, ramipril, perindopril, quinapril, moexcipril, and trandolapril. Examples of ARBs include losartan, candesartan, irbesartan, and valsartan. Appropriate beta-blockers include, but are not limited to, alprenolol, carteolol, levobunolol, mepindolol, metipranolol, nadolol, oxyprenolol, penbutrol, pindolol, propranolol, sotalol, timolol, acebutrol, atenolol, betaxolol, bisoprolol, esmolol, metoprolol, nebibolol, carvedilol, ceriprolol, labetalol, and butaxamine. Diuretics include, but are not limited to, calcium chloride, ammonium chloride, amphotericin B, lithium citrate, goldenrod, juniper, dopamine, acetazolamide, dorzolamide, bumetanide, ethacrine, furosemide, torsemide, glucose, mannitol, amiloride, spironolactone, triamterene, bendroflumethiazide, hydrochlorothiazide, caffeine, and theophylline. Examples of antiarrhythmic drugs include, but are not limited to, disopyramide, procainamide, quinidine, lidocaine, phenytoin, flecainide, propafenone, propranolol, timolol, metoprolol, sotalol, atenolol, amiodarone, sotalol, bretylium, verapamil, and diltiazem.Examples of aldosterones include, but are not limited to, spironolactone, eplerenone, canrenone, potassium canrenoate, and finerenone.

[0027] Treatment method: A second object of the present invention relates to a method for treating adverse post-ischemic cardiac remodeling in patients who have experienced myocardial infarction, comprising administering a therapeutically effective amount of a GDF3 inhibitor to a subject.

[0028] As used herein, the terms “treatment” or “to treat” include both preventive or prophylactic treatments, as well as curative or disease-modifying treatments, and include treatments for patients at risk of developing the disease or suspected of having the disease, and patients diagnosed as being ill or suffering from the disease or condition, and include the suppression of clinical relapses. Treatments are administered to subjects who have a medical disability or are likely to eventually acquire such disability in order to prevent, cure, delay the onset, reduce the severity of, or improve one or more symptoms of the disease or relapsing disease, or to extend the expected lifespan of the subject in the absence of such treatment. “Treatment regimen” means a pattern of treatment for the disease, e.g., a pattern of dosages used during treatment. A treatment regimen may include an induction regimen and a maintenance regimen. The phrase “induction regimen” or “induction period” means a treatment regimen (or part of a treatment regimen) used for the initial treatment of the disease. The general purpose of an induction regimen is to provide the patient with a high level of medication during the initial period of the treatment regimen. An introduction regimen may (partially or entirely) be a “loading regimen,” which includes administering a higher dose of the drug than the physician would use during the maintenance regimen, administering the drug more frequently than the physician would use during the maintenance regimen, or both. The phrase “maintenance regimen” or “maintenance period” refers to a treatment regimen (or part of a treatment regimen) used to maintain a patient during treatment for a disease, for example, to maintain the patient in remission for an extended period (several months or several years). A maintenance regimen may be continuous therapy (e.g., administering the drug at regular intervals, e.g., once a week, once a month, once a year) or intermittent therapy (e.g., interruption of treatment, intermittent treatment, treatment on relapse, or treatment when specific predetermined criteria (e.g., disease onset) are met).

[0029] Specifically, the method of the present invention is suitable for preventing or mitigating damage to the myocardium after myocardial infarction, after ischemia-reperfusion, during ischemia-reperfusion, or before ischemia-reperfusion. More specifically, the method of the present invention is particularly suitable for mitigating left ventricular remodeling after ischemia. More specifically, the method of the present invention is suitable for increasing the left ventricular ejection fraction (LVEF), and / or suppressing left ventricular enlargement, and / or decreasing left ventricular end-systolic volume, and / or suppressing left ventricular end-diastolic volume, and / or improving left ventricular dysfunction, and / or improving myocardial contractility.

[0030] As used herein, the term “GDF3 inhibitor” refers to any natural or non-natural compound capable of inhibiting the activity or expression of GDF3. This term encompasses any GDF3 inhibitor currently known or hereafter identified in the Art, and includes any chemical entity that, when administered to a patient, results in inhibition or downregulation of the biological activity or expression of GDF3.

[0031] In some embodiments, the GDF3 inhibitor is an anti-GDF3 neutralizing antibody. In some embodiments, the anti-GDF3 neutralizing antibody binds to the mature domain of GDF3. In some embodiments, the anti-GDF3 neutralizing antibody binds to an amino acid sequence ranging from amino acid residue at position 251 to amino acid residue at position 364 of SEQ ID NO: 1.

[0032] As used herein, the term “antibody” is used to refer to any antibody-like molecule having an antigen-binding domain, and this term includes antibody fragments containing the antigen-binding domain. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, which is specifically incorporated herein by reference). Specifically, for diabetes, further details are provided in European Patent No. 404,097 and International Publication No. 93 / 11161. For linear antibodies, further details are provided in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be produced by treating an antibody with pepsin. The resulting F(ab')2 fragments can be treated to reduce disulfide crosslinks to produce Fab' fragments. Digestion with papain can result in the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be synthesized by recombinant technology or chemically. Techniques for generating antibody fragments are well known in the art and are described, for example, in Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001; and Reiter et al., 1996, further describing and enabling the generation of effective antibody fragments.

[0033] As used herein, the term “neutralizing antibody” refers to an antibody capable of reducing or inhibiting (blocking) the activity or signaling of a ligand as measured by an in vivo assay or an in vitro assay.

[0034] In some embodiments, the antibodies of the present invention are single-domain antibodies. As used herein, the term “single-domain antibody” has a common meaning in the art and refers to a single heavy-chain variable domain of an antibody of a type that can be found in camelid mammals, which inherently lack a light chain. Such single-domain antibodies are also known as “nanobody” (trademark).

[0035] In some embodiments, the antibodies of the present invention are fully human antibodies. As used herein, the term “fully human” means, for example, an antibody or antibody fragment, or other immunoglobulin, whose entire molecule is of human origin or which consists of the same amino acid sequence as the human type of antibody or immunoglobulin. Fully human monoclonal antibodies can also be prepared by immunizing transgenic mice against most of the loci of human immunoglobulin heavy and light chains. See, for example, U.S. Patents 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and the references cited herein (their contents incorporated herein by reference).

[0036] In some embodiments, the antibodies of the present invention are humanized antibodies. As used herein, “humanized” means that some, most, or all of the amino acids outside the CDR region are substituted with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described herein by reference, in U.S. Patents 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205.

[0037] In some embodiments, GDF3 inhibitors are aptamers. Aptamers are a class of molecules that represent an alternative to an antibody in terms of molecular recognition. Aptamers are oligonucleotide sequences that have the ability to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through phylogenetic evolution of ligands (SELEX) by exponential enrichment of a random sequence library. A random sequence library can be obtained by combinatorial chemosynthesis of DNA. In this library, each member is a linear oligomer of a specific sequence, ultimately chemically modified. Peptide aptamers consist of a conformationally constrained antibody-variable region expressed by a platform protein such as E. coli thioredoxin A, selected from the combinatorial library by two hybrid methods (Colas et al., 1996).

[0038] In some embodiments, the GDF3 inhibitor is an inhibitor of GDF3 expression. “Expression inhibitor” refers to a natural or synthetic compound having a biological effect of inhibiting gene expression. In preferred embodiments of the present invention, the gene expression inhibitor is an siRNA, antisense oligonucleotide, or ribozyme. For example, antisense oligonucleotides, including antisense RNA and antisense DNA molecules, act to directly block the translation of GDF3 mRNA by binding to GDF3 mRNA, thereby preventing protein translation or increasing mRNA degradation, and thereby reducing the level of GDF3 in the cell, e.g., its activity. For example, antisense oligonucleotides of at least about 15 nucleotides that are complementary to a specific region of the mRNA transcription sequence encoding GDF3 can be synthesized, for example, by conventional phosphodiester techniques. Methods using antisense techniques to specifically inhibit the gene expression of genes whose sequences are publicly known are well known in the art (see, for example, U.S. Patents 6,566,135, 6,566,131, 6,365,354, 6,410,323, 6,107,091, 6,046,321; and 5,981,732). Small suppressor RNAs (siRNAs) can also function as expression inhibitors for use in the present invention. GDF3 gene expression can be reduced by contacting a patient or cells with small double-stranded RNA (dsRNA), or a vector or construct that induces the production of small double-stranded RNA, resulting in the specific inhibition of GDF3 gene expression (i.e., RNA interference or RNAi). The antisense oligonucleotides, siRNAs, shRNAs, or ribozyme nucleic acids of the present invention may be delivered in vivo, alone, or in conjunction with a vector. In its broadest sense, a “vector” is any vehicle capable of facilitating the transfer of antisense oligonucleotides, siRNAs, shRNAs, or ribozyme nucleic acids into cells, and typically cells expressing GDF3. Typically, a vector transports nucleic acids into cells with less degradation compared to the degree of degradation that would occur in the absence of the vector.Generally, vectors useful in the present invention include, but are not limited to, plasmids, phagemids, viruses, antisense oligonucleotides, siRNA, shRNA, or other vehicles derived from viral or bacterial sources manipulated by insertion or incorporation of ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to, nucleic acid sequences derived from RNA viruses such as: retroviruses such as Moloney's mouse leukemia virus, Harvey's mouse sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; adenoviruses, adeno-associated viruses; SV40 virus; polyomavirus; Epstein-Barr virus; papillomavirus; herpesvirus; vaccinia virus; poliovirus; and retroviruses. Other vectors known in the art but not specifically named can readily be used. In some embodiments, the inhibitor of expression is an endonuclease. The term “endonuclease” refers to an enzyme that cleaves phosphodiester bonds within a polynucleotide chain. Some endonucleases, like deoxyribonuclease I, cleave DNA relatively nonspecifically (regardless of sequence), while many, typically called restriction endonucleases or restriction enzymes, cleave only at very specific nucleotide sequences. The mechanisms behind endonuclease-based genome inactivation generally require a first step of DNA single-strand or double-strand breaks, which can then be used for DNA inactivation, triggering two distinct cellular mechanisms for DNA repair: error-prone non-homologous end joining (NHEJ) and high-fidelity homology-directed repair (HDR). In certain embodiments, the endonuclease is CRISPR-cas. As used herein, the term "CRISPR-cas" has a general meaning in the art and refers to clustered, regularly spaced short repeats associated with segments of prokaryotic DNA containing short repeats of a nucleotide sequence. In certain embodiments, the endonuclease is CRISPR-cas9 derived from Streptococcus pyogenes. The CRISPR / Cas9 system is described in U.S. Patent Nos. 8,697,359,B1 and 2014 / 0068,797.In one embodiment, the endonuclease is CRISPR-Cpf1, a more recently characterized CRISPR derived from Provotella and Francisella 1 (Cpf1) as described by Zetsche et al. ("Cpf1 is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13").

[0039] In some embodiments, a GDF3 inhibitor is administered to a subject having one or more signs or symptoms of acute myocardial infarction injury. In some embodiments, the subject has one or more signs or symptoms of myocardial infarction, such as chest pain described as pressure, fullness, or tightness in the middle of the chest; chest pain radiating to the jaw, teeth, shoulders, arms, and / or back; shortness of breath or difficulty breathing; epigastric discomfort with or without nausea and vomiting; and diaphoresis or sweating.

[0040] In some embodiments, the GDF3 inhibitor is administered to the subject simultaneously with or sequentially (i.e., before or after) the revascularization procedure performed. In some embodiments, the subject receives the GDF3 inhibitor before, during, and after the revascularization procedure. In some embodiments, the subject receives the GDF3 inhibitor as a bolus dose immediately before the revascularization procedure. In some embodiments, the subject receives the GDF3 inhibitor continuously during and after the revascularization procedure. In some embodiments, the subject receives the GDF3 inhibitor at a time selected from the group consisting of at least 3 hours after the revascularization procedure; at least 5 hours after the revascularization procedure; at least 8 hours after the revascularization procedure; at least 12 hours after the revascularization procedure; and at least 24 hours after the revascularization procedure. In some embodiments, the revascularization procedure is selected from the group consisting of percutaneous coronary intervention; balloon angioplasty; bypass graft insertion; stent insertion; directional coronary atherosclerosis; procedures with one or more thrombolytic agents; and occlusion removal.

[0041] "Therapeutally effective dose" means an amount of the active ingredient sufficient to treat or alleviate symptoms with a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily dose of the compounds and compositions of the present invention will be determined by the attending physician within the bounds of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disorder being treated and its severity; the activity of the particular compound used; the particular composition used, the subject's age, weight, general health, sex, and diet; the timing of administration, route of administration, and excretion rate of the particular compound used; the duration of treatment; drugs used in combination with the active ingredient; and factors similar to those well known in the medical field. For example, starting administration of the compound at a level lower than necessary to achieve the desired therapeutic effect and gradually increasing the dose until the desired effect is achieved is well within the scope of the art. However, the daily dose of the product may vary over a wide range from 0.01 mg to 1,000 mg per day for adults. Typically, the composition contains 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the active ingredient for symptomatic adjustment of the dose to the subject being treated. The drug typically contains about 0.01 mg to about 500 mg of the active ingredient, and typically 1 mg to about 100 mg of the active ingredient. Usually, the effective dose of the drug is supplied at a dose level of about 0.0002 mg to about 20 mg per day of body weight, and especially about 0.001 mg to 7 mg per kg of body weight per day.

[0042] Typically, the active ingredient of the present invention (e.g., a GDF3 inhibitor) is combined with a pharmaceutically acceptable excipient and optionally a sustained-release matrix such as a biodegradable polymer to form a pharmaceutical composition. The terms “pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that, as necessary, do not produce adverse reactions, allergies, or other undesirable reactions when administered to mammals, particularly humans. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid. The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of a coating agent such as lecithin, by maintaining the required particle size in the case of a dispersant, and by the use of a surfactant. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Long-lasting absorption of the injectable composition is achieved by the use of absorption retardants in the composition, such as aluminum monostearate and gelatin. In the pharmaceutical composition of the present invention, the active ingredient of the present invention can be administered in unit dose forms as a mixture with a conventional pharmaceutical support. Suitable unit dose forms include oral route forms such as tablets, gel capsules, powders, granules, and oral suspensions or solutions; sublingual and oral administration forms; aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, intrathecal, and intranasal administration forms; and rectal administration forms. [Brief explanation of the drawing]

[0043] The present invention is further illustrated by the following figures and embodiments. However, these embodiments and figures should not be construed in any way that limits the scope of the present invention. [Figure 1a]GDF3 is a circulating factor secreted after myocardial infarction and may predict adverse cardiac remodeling in humans. a. Representative Western blot and quantification of mature GDF3 in patients with non-incomplete (NF) (n=6) and incomplete (HF) (n=9). *P<0.05 determined by Mann-Whitney test. [Figure 1b] GDF3 is a circulating factor secreted after myocardial infarction and may predict adverse cardiac remodeling in humans. b. GDF3 levels *P=0.05 in non-remodelers (n=24) and remodelers (n=56) four days post-myocardial infarction were analyzed by the Mann-Whitney nonparametric t-test. [Figure 1c] GDF3 is a circulating factor secreted after myocardial infarction and may predict adverse cardiac remodeling in humans. c. ROC curve for distinguishing between remodelers and non-remodelers. [Figure 1d] GDF3 is a circulating factor secreted after myocardial infarction and may predict adverse cardiac remodeling in humans. d-g. LVEDVi (d), LVEF (e), infarct size (f), and number of akinetic segments (g) at 6 months after MI in patients from the low GDF3 group (<1375 pg / mL) and the high GDF3 group (>1375 pg / mL). *Mann-Whitney nonparametric t-test* P<0.05, **P<0.01. [Figure 1e] GDF3 is a circulating factor secreted after myocardial infarction and may predict adverse cardiac remodeling in humans. d-g. LVEDVi (d), LVEF (e), infarct size (f), and number of akinetic segments (g) at 6 months after MI in patients from the low GDF3 group (<1375 pg / mL) and the high GDF3 group (>1375 pg / mL). *Mann-Whitney nonparametric t-test* P<0.05, **P<0.01. [Figure 1f]GDF3 is a circulating factor secreted after myocardial infarction and may predict adverse cardiac remodeling in humans. d-g. LVEDVi (d), LVEF (e), infarct size (f), and number of akinetic segments (g) at 6 months after MI in patients from the low GDF3 group (<1375 pg / mL) and the high GDF3 group (>1375 pg / mL). *Mann-Whitney nonparametric t-test* P<0.05, **P<0.01. [Figure 1g] GDF3 is a circulating factor secreted after myocardial infarction and may predict adverse cardiac remodeling in humans. d-g. LVEDVi (d), LVEF (e), infarct size (f), and number of akinetic segments (g) at 6 months after MI in patients from the low GDF3 group (<1375 pg / mL) and the high GDF3 group (>1375 pg / mL). *Mann-Whitney nonparametric t-test* P<0.05, **P<0.01. [Examples]

[0044] Materials and methods All procedures and animal care protocols were approved by our institutional research committee (CEEA34 and French ministry of research, N° 2019050221153452) and complied with the animal care guidelines of the European Parliament Directive 2010 / 63 / EU. All animals received humane care in accordance with the "Principles of the Management of Laboratory Animals" formulated by the National Association for Medical Research and the "Guidelines on the Management and Use of Laboratory Animals" prepared by the Laboratory Animal Resources Institute and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1996).

[0045] animal research design 8-week-old or 13-week-old male C57BL / 6J and PW1 reporter (PW1 nLacZMice were anesthetized in an induction chamber containing 2% isoflurane mixed with 1.0 L / min of 100% O2, and placed supine on a heating pad to maintain body temperature. Endotracheal tubes were inserted into the mice, and then connected to a rodent ventilator (180 breaths per minute, 200 μL tidal volume). Anesthesia was maintained throughout the surgical procedure with 1.5–2% isoflurane and O2. The pericardium was incised by accessing the chest from the left side through the intercostal space. The LAD was exposed and surrounded proximal with 8.0 prolene sutures. The sutures were briefly snred, and the arterial area was branched to confirm ligation. Mice were analyzed 7 days after permanent LAD ligation. Blood samples were collected in heparin-coated Eppendorf tubes, immediately centrifuged at 200 × g at 4°C for 15 minutes to separate the plasma, and stored at -80°C until analysis. The heart was resected and immediately digested for FACS sorting or qPCR analysis.

[0046] Cell isolation and fluorescence-activated cell sorting (FACS) PW1 + CD51 + As described above, cardiac cells were sorted. 29 Simply put, the small cell suspension is prepared using 8-week-old PW1 cells. nLacZ Cells were prepared from the whole heart when the atria were removed from mice. The ventricles were enzymatically digested and dissociated using collagenase II. The following antibodies were used for cell sorting: BUV737-tagged anti-CD31 (1:100 dilution; BD Bioscience), BUV395-tagged anti-TER119 (1:50 dilution, BD Biosciences), and phycoerythrin-cyanine 7-tagged anti-CD45 (1:500 dilution, eBioscience). To detect β-gal reporter activity, cells were treated with the fluorescent substrate 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C). 12 The samples were incubated with FDG at 37°C for 1 hour. Various populations were gated using a FACSAria II cytometer (BD Biosciences), analyzed, and selected.

[0047] CyQUANT® Cell Proliferation Assay PW1 selected by FACS + and PW1 - (FDG - The cells were seeded in 24-well plates at a density of 15,000 cells per well and cultured for 5 days under normal conditions in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS, Sigma) and 1% penicillin and streptomycin (Sigma). The medium was collected and used to incubate serum-starved MEFs (cultured for 24 hours under normal conditions, then cultured for 24 hours under serum-starved conditions) for 24 hours. MEF growth was evaluated using the CyQUANT cell proliferation assay according to the manufacturer's instructions. MEFs cultured in complete medium were used as a control.

[0048] RNA sequencing and bioinformatics analysis Libraries were prepared using a total of 300 ng of total RNA extracted from cells immediately isolated using the SureSelectStrand-SpecificRNA Kit (Agilent), according to the manufacturer's instructions. The resulting libraries were quality-checked and quantified by peak merging on a Bioanalyzer Highsensitivity DNA labchip (Agilent). Equal-volume pools of 12 purified libraries were prepared, and each library was tagged with a different index. The mRNA pool libraries were finally sequenced on an Illumina Hiseq 1500 instrument using a rapid flow cell. The pools were loaded into two lanes of the flow cell. 2 × 100 bp paired-end sequencing was performed.

[0049] The Illumina filter discards the reads that did not pass through, and the Cutadapt program 30 After trimming low-quality sequenced bases (q<) using ENSEMBL, downstream analysis was limited to readouts longer than 90 bp. Selected readouts were then analyzed against the complete mouse reference genome and ENSEMBL. 32RSEM package from GTF transcript annotation file 31 The transcripts were mapped to a mouse reference transcriptome generated by [the specified method]. The RSEM program was used to align and estimate the abundance of each transcript from 12 processed samples. Transcripts counted as greater than 10 in more than two samples (N=36,948) were considered expressed and retained for further analysis. The abundances of transcripts assigned to the same gene were combined to provide profiling for 16,403 genes. Analysis was performed in the R environment (version 3.2.2).

[0050] A Galaxy 15.10 instance was installed locally on the server machine. WolfPsort, TMHMM, and SignalP were obtained from the CBS prediction server (https: / / services.healthtech.dtu.dk / , accessed April 15, 2020). Nuclear localization signals were determined using NLStradamus and PredictNLS in parallel. Each dataset from RNA sequences corresponding to different populations was then processed through a pipeline designed to select sequences containing signal peptides, transmembrane segments, sequences without nuclear localization signals, and sequences containing structural features consistent with active secretion via classical or non-classical secretory pathways.

[0051] qPCR analysis RNA was extracted from cardiac cells isolated 7 days after surgery from MI mice and SHAMC57BL / 6J mice using the RNAqueous Micro Kit (AM1931, Invitrogen), following the manufacturer's instructions. Next, 500 ng of the extracted RNA was subjected to reverse transcription using the SuperScript IVVILO Kit (11756050, Invitrogen), following the manufacturer's instructions. The resulting cDNA was subjected to qPCR using SYBR Select Master Mix (4472908, Applied Biosystems) on a Quant Studio 3 Real-Time PCR system (Thermo Fisher) under the following conditions: 10 minutes at 95°C, 15 seconds at 95°C and 1 minute at 60°C for 40 cycles, followed by 10 seconds at 95°C and 1 minute at 60°C. Target gene expression was normalized to RPL13 expression, and then -2 ΔΔCT The analysis was performed using a specific method.

[0052] Culture and transfection of HEK293 cells HEK293 cells were cultured at 37°C in DMEM GlutaMax™-1 (Life Technologies) supplemented with 10% FBS, 1% penicillin, and streptomycin, in the presence of 5% CO2. HEK293 cells were then placed in 6-well plates in antibiotic-free medium, with 6 × 10⁶ cells per plate. 5 Cells were plated. After 24 hours, transfection with expression plasmids (Origene and Genscript) using lipofectamine (trademark) 2000 (Life Technologies) was performed using 2 μg of plasmid and 6 μL of lipofectamine (trademark) 2000 diluted with Opti-MEM (Life Technologies), according to the manufacturer's protocol. Cells were cultured for 2 days, then serum was starved for 8 hours, after which conditioned media was collected and centrifuged at 200 × g for 10 minutes. The supernatant was stored at -80°C until MEF treatment.

[0053] Isolation of mouse embryonic fibroblasts Primary MEFs were isolated from C57bL / 6J mouse embryos 13.5 days post-mating. Pregnant females were euthanized by cervical luxation, and the embryos were surgically removed and isolated from maternal tissue and yolk sac in ice-cold phosphate-buffered saline (PBS). Subsequently, the embryos were decapitated and the internal organs removed (removal of heart, spleen, liver, and intestines). The bodies were washed with ice-cold PBS to remove blood, and then finely ground in a petri dish without PBS. The samples were incubated at 37°C for 15 minutes in a digestion solution (0.05% trypsin-EDTA solution (Life Technologies), 0.1 mg / mL DNase1 (Sigma)). The suspension was allowed to settle. The supernatant was drained and mixed with MEF medium (DMEM (4.5 g / L D-glucose) (Life Technologies), 10% FBS, 1% penicillin-streptomycin, 1% non-essential amino acids (Life Technologies)), and centrifuged at 200 × g for 5 minutes. After centrifugation, the pellet containing MEF was resuspended in MEF medium. The pellet from tissue digestion was resuspended in the digestion solution and incubated at 37°C for 15 minutes. The cells were allowed to settle, the supernatant was drained, and processed as described above. Cells from the first and second digestion stages were pooled and plated in petri dishes. Each petri dish received a cell suspension equivalent to 1.5 embryos.

[0054] After 12 hours, the medium was changed to remove non-adherent cells and debris. MEFs were subculturized when they reached 80% confluence. MEFs were harvested by trypsin treatment, centrifuged, and resuspended in a freezing medium (DMEM 4.5 g / L D-glucose, 1% penicillin-streptomycin, 10% dimethyl sulfoxide). Primary MEFs were cultured for subculturing 0 to 4.

[0055] Western blot analysis Proteins were extracted from frozen mouse heart tissue using a Dounce-Potter homogenizer and placed in ice-cold radioimmunoprecipitation (RIPA) buffer (50 mM Tris pH 7.4, 150 mM sodium chloride, 1% IGEPARCA-630, 50 mM deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)) supplemented with 1% antiprotease (Sigma-Aldrich), 1% antiphosphatase inhibitors (phosphatase inhibitor cocktails 2 and 3, Sigma-Aldrich), and 1 mM sodium orthovanadate. After incubation at 4°C for 1 hour, the mixture was homogenized at 15,300 × g, centrifuged at 4°C for 15 minutes, and the supernatant containing the proteins was collected. Cardiac PW1 samples were taken from 22 mice. + Cells and PW1 from 16 mice - Cells were pooled, centrifuged at 500×g at 4°C for 15 minutes, and dissolved in urea-thiourea buffer (5M urea, 2M thiourea, 50mM dithiothreitol (DTT), PBS H7.4 containing 0.1% SDS). Proteins were extracted as described above. Protein concentrations in all samples were determined using a Bradford-based protein assay (Bio-Rad).

[0056] As mentioned above 7After separating cardiomyocytes and non-cardiomyocytes derived from adult mouse hearts, the proteins were loaded onto NuPAGE® Novex® 4-12% Bis-Tris gel (Life Technologies) and denatured at 70°C for 10 minutes. After 3 hours of electrophoresis, the proteins were transferred onto a nitrocellulose membrane using a Trans-Blot Turbo Transfer System (Bio-Rad) and stained with 0.1% Ponceau S (w / v in 5% acetic acid) to evaluate transfer quality and uniform loading. The membranes were blocked for 1 hour with constant shaking in 0.1% Tween-20 supplemented Tris-buffered saline (TBS-Tween) containing 5% skim milk. Next, they were incubated overnight at 4°C with primary antibodies specific to GDF3 (1:1000 for tissue and 1:500 for plasma, Abcam) and FLAG (1:1000, Sigma-Aldrich), diluted in 5% skim milk / TBS-Tween. After washing, the membranes were incubated for 1 hour at room temperature (23°C) with secondary antibodies conjugated with wasabi peroxidase (HRP) diluted in 5% skim milk / TBS-Tween. The membranes were then washed and incubated for 5 minutes with Super Signal® West Pico PLUS Chemiluminescent Substrate (Life Technologies). The images were then captured using a Chemidoc® XRS+ camera (Bio-Rad) and analyzed using ImageLab® software.

[0057] ELISA GDF3 levels were measured by a GDF3 sandwich ELISA assay (GenWay, GWB-KBBHW6) according to the manufacturer's instructions. Briefly, the standard and diluted sample (1:16 with standard diluent) were added to an anti-GDF3 microplate (a plate pre-coated with a GDF3-specific antibody) and incubated at 37°C for 1 hour. After removing the standard and sample, biotinylated GDF3 detection antibody was applied. The plate was incubated at 37°C for 1 hour. The wells were washed and then incubated with avidin-HRP conjugate at 37°C for 30 minutes. Finally, after thorough washing, the wells were incubated with 3,5,3',5'-tetramethylbenzidine (TMB) substrate in the dark at 37°C for 15 minutes. The blue product due to oxidation of the TMB substrate turned yellow after incubation at 37°C for 15 minutes following the completion of the reaction by adding stop solution. The absorbance at 450 nm was quantitatively proportional to the amount of GDF3 incorporated into the well and was measured using a microplate reader.

[0058] Sub-study of PREGICA cardiac MRI We used a plasma bank (Predisposition Genetical in Cardiac Insufficiency, clinicaltrials.gov identifier NCT01113268) from 80 patients enrolled in a secondary study of expected PREGICA cardiac MRI in first-time STEMI patients. Details of this study are already described in (Garcia R, Bouleti C, Sirol M et al. VEGF-A plasma levels are associated with microvascular obstruction in patients with ST-segment elevation myocardial infarction. Int J Cardiol). (2019;291:19-24). Briefly, this study involved six cardiac centers in France and enrolled patients aged 18 to 80 years with a first-time STEMI referenced between 2010 and 2017 who presented within 24 hours of symptom onset. STEMI was defined by the presence of ST segment elevation on ECG, a significant elevation of troponin (more than 3 times the upper limit of normal), and the presence of at least three akinetic LV segments on the first transthoracic echocardiogram. Patients with a diagnosis of persistent atrial fibrillation, a history of MI, or a history of heart disease were not included. All patients underwent coronary angiography and primary PCI within the first 24 hours. In a subset of patients defined for the secondary study of CMR in the PREGICA cohort... Cardiac MRI was performed using 1.5T units at 4±2 days post-admission and at 6 months follow-up. A standardized MRI protocol was followed across all centers, and images were intensively analyzed. Cine images were acquired in long-axis and short-axis views using a breath-hold steady-state free precession sequence. Left ventricular (LV) volume and ejection fraction (EF) were derived using a stack of short-axis slices covering from the atrioventricular ring to the apex. Late gadolinium-enhanced (LGE) images were acquired in the same long-axis and short-axis views as the cine images, 10 minutes after intravenous injection of a gadolinium-based contrast agent, using a breath-hold segmented T1-weighted inversion recovery gradient echo sequence. LGE images were evaluated by infarct size. Blood samples were collected concurrently with cardiac MRI.GDF3 was quantified using an ELISA assay in available plasma collected on day 4 (n=80). This study was approved by the institutional review board, and all patients provided written informed consent.

[0059] statistical analysis Mouse and in vitro studies. The sample size (n) used in each experiment is recorded in the legends of the text and figures. All experiments were conducted at least twice independently. Data are expressed as mean ± standard deviation (SD). Quantitative data were analyzed using pairwise comparisons with one-way analysis of variance (ANOVA, Kruskal-Wallis test) and Dunnett's post-hoc test for multiple comparisons. The Mann-Whitney U test was used to compare continuous variables between two groups.

[0060] Analysis of a secondary study of PREGICA cardiac MRI. P-values ​​were obtained from the chi-squared test statistic for the binomial variable and using the Mann-Whitney U test to compare continuous variables between the two groups. The association between GDF3 levels and the likelihood of adverse cardiac remodeling was assessed using a linear regression model with additional adjustments for age and sex.

[0061] All statistical analyses were performed using GraphPadPrism 8.0 (GraphPad Software, Inc., San Diego, CA). Values ​​P<0.05 indicate statistical significance.

[0062] result PW1 derived from ischemic heart + The cells release factors that promote the proliferation of fibroblasts. Adult PW1 nLacZ As mentioned above, the reporter mouse 6,7 Ischemic cardiac injury was induced by ligation of the left anterior descending coronary artery (LAD). Seven days after injury, the hearts were harvested from MI mice in the same manner as in SHAM-operated mice, and PW1 was identified by fluorescence-activated cell sorting (FACS). +Cells were isolated (not shown). After 5 days of culture, conditional media were collected from these cells and used to culture mouse embryonic fibroblasts (MEFs) for 24 hours (not shown). The effect of the conditional media on MEF proliferation was evaluated using the CyQUANT® cell proliferation assay. The results of the cell proliferation assay were compared with conditional media derived from control cells or PW1 cells derived from SHAM-modified hearts. + PW1 isolated from ischemic heart compared to those treated with cells + A significant increase in the proliferation of MEFs incubated in cell-derived conditional media was revealed (not shown). PW1 - There was no significant increase corresponding to the cell-derived condition medium (not shown). These observations suggest that activated PW1 derived from ischemic heart + This suggests that cells may release growth-promoting factors, which can induce the proliferation of resident fibroblasts.

[0063] RNA sequencing (RNA-seq) and bioinformatics analysis predict candidate biomarkers involved in paracrine activity. FACS isolation PW1 derived from SHAM and MI mice. + The cell transcriptome is characterized by RNA-seq, and PW1 + The effect of the ischemic cardiac environment on cellular paracrine potentials was investigated. RNA-seq output files were filtered, sorted, and quality-controlled to obtain a list of transcripts showing the highest signal intensity (not shown). Cardiac PW1 + Comparative analyses were performed to understand disease-induced changes in the secretory behavior of final candidates that showed more than twice the expression level in ischemic conditions compared to normal conditions (not shown). Subsequently, a series of bioinformatics algorithms were used to test the predicted amino acid sequences of the corresponding genes and identify secretory proteins. Proteins that possess a predicted N-terminal endoplasmic reticulum (ER) targeting signal peptide but lack the predicted transmembrane domain and intracellular localization signal (i.e., lacking ER retention signal, mitochondrial target peptide, or nuclear export signal) were examined (not shown). Progressive filtering was performed under ischemic conditions to identify cardiac PW1 +We provided a total of 24 secretory proteins overexpressed by cells (not shown). Next, quantitative polymerase chain reaction (qPCR) confirmed that the expression of 12 of these 24 candidates was significantly increased in ischemic hearts (distant or infarcted areas) compared to normal hearts (not shown).

[0064] Compared to the secretome of control cells, PW1 activated in MI + The cellular secretome included several growth factors, cytokines, and enzymes, as well as several factors whose properties are not yet fully understood (not shown). Secretion of cytokines such as the growth factors GDF3, NDP, and CCL8, and enzymes such as CELA1 and PRTN3 was more than twice as high in MI hearts than in SHAM hearts (not shown).

[0065] Transfection experiments will identify candidate cells that have a proliferative effect on fibroblasts. Next, we investigated the effects of candidate proteins on the proliferation of cultured embryonic fibroblasts and adult cardiac fibroblasts. Based on the biological function of their gene ontologies, we selected six candidates (CCL8, CELA1, GDF3, NDP, PRNT3, PROK2) that were potentially associated with cell proliferation. Conversely, we excluded lipoproteins APOC2, APOC4, and SAA3, coagulation factor F10, and the less characterized C1QTNF3 and DMKN. The cDNAs of these six proteins were separately cloned into mammalian expression plasmids, which were then used to introduce them into HEK-293 cells (not shown). FLAG epitope-tagged fibroblast growth factor 23 cDNA was used as a positive control, while an empty vector served as a negative control. Forty-eight hours after transfection, the conditional medium was collected and tested to confirm overexpression of secreted proteins (not shown), and then used to incubate serum-starved MEFs. Evaluation of cell proliferation rates after 24 hours of treatment revealed four factors that significantly induced MEF proliferation compared to the control treatment: growth differentiation factor-3 (GDF3), norincysteine ​​knot growth factor (NDP), prokinethicin 2 (PROK2), and chymotrypsin-like elastase family member 1 (CELA1) (not shown). The proliferation effects of three of these four candidates (GDF3, NDP, and PROK2) were further confirmed using newly isolated adult cardiac fibroblasts (not shown). Therefore, the cell proliferation assay facilitated the selection of three candidate cells, GDF3, NDP, and PROK2, from 12 overexpression markers.

[0066] Of these remaining three candidates, GDF3 (also known as Vg-related gene 2) exhibited both the most significant overexpression in ischemic hearts and one of the most important increases in fibroblast proliferation. GDF3 is a member of the TGF-β superfamily, consisting of 366 amino acid residues. Human and mouse GDF3 exhibits 76.6% nucleotide homology and 69.3% peptide identity. 12The predicted amino acid sequence includes a signaling sequence for secretion at the hydrophobic NH2 terminus, a prodomain that facilitates cysteine-mediated disulfide bond formation with another family member, and a proteolytic processing site (not shown) estimated to be 114 amino acid residues long. Cleavage of GDF3 at this residue produces a mature GDF3 protein with a length of 114 residues. 11 GDF3 plays an important role in early development in mice and humans. 13 Its expression is less frequent in adult organs, and is particularly negligible in the adult heart. 10、14、15 While the function and implications of GDF3 in adult hearts remain unclear, the biological function of GO suggests involvement with the SMAD protein signaling pathway, which is highly relevant to the process of cardiac fibrosis. This is consistent with the highest proliferative effect of GDF3 among all candidates for MEF (not shown). Transfection experiments (not shown) confirmed that GDF3 is a secreted protein, as indicated by a full-length protein band on the supernatant Western blot (not shown). These observations together suggest a potential role of GDF3 in regulating fibroblast proliferation in scar tissue and spurred investigation of GDF3 expression profiles in mouse and human MI hearts.

[0067] GDF3 levels increase in the plasma and infarcted areas of mouse hearts after MI. Based on transcriptome results, we attempted to evaluate GDF3 expression throughout the heart and determine its cell source by Western blotting. Mature forms of GDF3 were detected in both neonatal and normal adult hearts (not shown). Specifically, in adult hearts, GDF3 was expressed only in the non-cardiac cell fraction and not in cardiomyocytes (not shown). Further analysis of the non-cardiac cell fraction revealed that specific expression of GDF3 was associated with PW1 + It was confirmed in cells, but PW1 in a normal heart -It was not observed in cell populations (not shown). To investigate the dysregulation of GDF3 expression in mouse hearts after MI, a permanent LAD mouse model was created and the heart was resected after 7 days. GDF3 expression in the infarcted area corresponding to scar tissue and the distant area were analyzed separately. Western blotting confirmed that GDF3 expression in the infarcted area of ​​MI hearts was higher than that in the corresponding area of ​​SHAM hearts (not shown). This result is consistent with our previous observations and the cardiac PW1 corresponding to MI. + This is consistent with the fate of cellular fibrosis, indicating that GDF3 is produced at the infarct site and suggesting its involvement in the post-MI scarring process.

[0068] GDF3 is a circulating factor secreted after MI (Minor Invasion). Considering the nature of this protein's secretion, we investigated whether free GDF3 could be detected in circulation by analyzing plasma samples from MI mice and SHAM mice. Similarly, Western blot analysis confirmed that MI mouse plasma contained higher levels of mature GDF3 than SHAM mouse plasma (not shown). We performed a GDF3-specific enzyme-linked immunosorbent assay (ELISA) to investigate the temporal changes in circulating levels of GDF3 in mouse plasma. Secreted protein levels increased from day 0 to day 2 post-MI, and then decreased until day 7.

[0069] Overall, and the heart PW1 + In line with the fate of cellular fibrillation, these results suggest that GDF3 may be a novel cardiokine secreted by these cells that could be associated with adverse cardiac remodeling after MI.

[0070] Circulating GDF3 levels serve as a marker of adverse remodeling after MI in humans. To investigate the clinical relevance of our findings in a mouse MI model, we first evaluated GDF3 expression in left ventricular tissue samples taken from both ischemic and non-ischemic hearts of patients. Western blot analysis revealed that GDF3 expression was stronger in ischemic hearts than in non-ischemic hearts (Figure 1a), indicating that the upregulation of GDF3 expression levels in the heart is a conserved response to MI.

[0071] Next, we investigated whether elevated circulating GDF3 levels could be associated with adverse cardiac remodeling after MI. We analyzed circulating GDF3 levels in 80 patients (PREGICA patient collection, NCT01113268) who presented within 24 hours of symptom onset and underwent primary percutaneous coronary intervention (PCI) for their first acute ST-elevation myocardial infarction (STEMI). Patients underwent initial clinical and biological evaluations on day 4 and serial magnetic resonance imaging (MRI) 4 days and 6 months after angioplasty. Detailed inclusion / exclusion criteria are described at https: / / clinicaltrials.gov / ct2 / show / NCT01113268. Baseline characteristics of these patients are shown in Table 1.

[0072] First, compared to the initial assessment using cMRI, the left ventricular end-diastolic volume (LVEDV) (LVEDVi, mL / m²) indexed against body surface area at 6 months was compared. 2Adverse cardiac remodeling was defined as an increase of 20% or more in GDF3. Accordingly, patients were classified into remodelers (n=24) and non-remodelers (n=56). GDF3 measured 4 days post-PCI was detectable in the plasma of these patients, and levels were significantly higher in remodelers than in non-remodelers (1364±521 vs. 1090±532 pg / mL, p=0.033) (Figure 1b). An increase of one standard deviation (SD) in GDF3 levels, after adjusting for age and sex, was associated with an increased risk of adverse remodeling (odds ratio (OR) = 1.76 [1.03~3.00], p=0.037). Plasma GDF3 levels did not show any statistically significant differences based on sex, smoking, or a history of hypertension and diabetes (p>0.10). Notably, even though these correlations did not reach statistically significant levels (p=0.28 and p=0.12, respectively), GDF3 levels were moderately correlated with CRP (p=0.13) and Hb1AC (p=0.18). To better assess the relationship between cardiac remodeling and plasma GDF3 levels, we first divided patients into four quartiles based on their GDF3 levels (measured 4 days post-MI), and compared LVEDVi and LVEF, measured by cardiac MRI 6 months post-MI, within these quartiles. We found that patients with the highest GDF3 levels (i.e., quartile 4) had significantly higher LVEDVi and lower LVEF compared to patients with lower GDF3 levels. Next, we performed receiver operating characteristic (ROC) curve analysis to determine if GDF3 levels could help distinguish between the two groups. There was a significant difference in the area under the ROC curve of the age and sex-adjusted model (0.69 [0.56~0.82] (p=0.05)), with a likelihood ratio of 2.154, sensitivity and specificity of 50% and 77%, respectively, and a cutoff value of 1375 pg / mL was calculated (Figure 1c).

[0073] Next, patients were classified according to this cutoff value (high GDF3 was defined as ≥1375 pg / mL, n=25, and low GDF3 was defined as <1375 pg / mL, n=55). Table 2 reports the main characteristics and cardiac MRI findings at baseline and 6 months post-MI for both groups. There was no significant imbalance in major cardiovascular risk factors between the two groups. Patients with high GDF3 levels (P<0.05) had a significantly longer delay from symptom onset to coronary artery occlusion (up to 1.2 hours). However, the peak of troponin, a surrogate marker for myocardial necrosis, was significantly lower in the high GDF3 group. Regarding cardiac remodeling, patients with high GDF3 did not show a significantly greater tendency towards greater cardiac dilation at 4 days post-MI compared to the low GDF3 group. However, in patients with high GDF3 levels, LVEDVi values ​​at 6 months post-MI were significantly higher (P<0.005) (Figure 1d) and abnormal (normal value <82 mL / m²). 2 These patients exhibited adverse cardiac remodeling associated with the progression of diastole. They also showed a significant decrease in LVEF at day 4 and month 6 (p<0.05), suggesting reduced recovery of systolic function after MI in these patients (Figure 1e). While there was no significant difference in total infarct size (whole heart weight %) between the two groups (Figure 1f), patients with high GDF3 levels had a higher proportion of akinetic segments on cardiac MRI at month 6 compared to patients with low GDF3 levels (Figure 1g). This result indicates a more significant pathological alteration to non-contractile scarring in the infarct area in patients with high GDF3 levels, highlighting its diagnostic importance as a marker of adverse cardiac remodeling after MI.

[0074] Consideration: Acute myocardial infarction characterized by left ventricular remodeling may progress towards the development of heart failure. 16 Markers that reflect myocardial damage may not be able to predict long-term left ventricular remodeling (troponin and creatine kinase), or may suffer from insufficient clinical data (galectin-3 and soluble interleukin-1 receptor-like markers). 17、18For example, galectin-3 has been shown to be involved in fibrosis and inflammation, and to be independently associated with the development of peripheral artery disease, in observational studies that included only Caucasians and Black individuals and did not exclude the influence of confounding factors. Therefore, in order to identify patients at high risk of HF and to provide timely disease management, it is essential to discover potential manufacturers that provide information on preclinical HF.

[0075] In order to contribute to wound healing, the cardiac extracorporeal membrane (ECM) undergoes constant remodeling upon injury. 19 Interestingly, the concept of ECM regulation via key molecules involved in intercellular communication has only recently become clear. This specification discusses cardiac PW1, a cell subpopulation skeptical of integrating repair processes in tissues including the heart. + cell 6、7 This study focused on these PW1 in ischemic mouse hearts. + We investigated the major differences in the secretome of interstitial cells. Notably, cardiac PW1 isolated from ischemic mouse hearts. + The growth-promoting effect observed in the conditional culture medium from cells was observed in cardiac PW1 isolated from normal mouse hearts. + Not only cells but also heart PW1 - It was not observed in cells either. RNA sequencing and bioinformatics analysis revealed several factors in MI heart (MI activated heart PW1) + Upregulation of the expression of 12 cellular secretory factors, specifically GDF3, PROK2, and NDP, was confirmed, and qPCR validation experiments (not shown) confirmed approximately 7-fold, 3-fold, and 3-fold upregulation of expression after MI. Furthermore, most of the 12 candidate dysregulation markers confirmed by qPCR validation are enriched in GO biological processes such as angiogenesis, inflammation, chemotaxis, and proliferation, and therefore, PW1 against MI + An important response from the cell population was observed.

[0076] MI is characterized by an acute inflammatory response involved in myocardial repair. 20However, uncontrolled chronic inflammation can lead to excessive damage and fibrosis, ultimately resulting in loss of cardiac function. 21 Cardiac inflammation and endothelial dysregulation are associated with extracellular matrix (ECM) remodeling. 22 And the TGF-β pathway has been consistently highlighted as a key molecular mediator in cardiac fibrosis. 23、24 .

[0077] GDF3 was initially shown to be involved in early embryonic development, muscle development, adipose tissue homeostasis, and energy balance as a member of the TGF-β superfamily, through the interaction of activin receptor-like kinase type I receptor B (ACVR1B, ALK4) and ACVR1C (ALK7) receptors. 25 Recent studies have highlighted the crucial role of GDF3 in macrophage function and the inflammatory cascade. Wang et al. recently described the role of GDF3 in macrophage polarization and endotoxin / septicement-induced cardiac injury. 26 This study identified a previously unrecognized function of GDF3 in cardiac fibrosis and demonstrated the dynamic changes in GDF3 levels in the blood and heart of mice and humans after MI.

[0078] This is the first report to investigate the potential prognostic role of GDF3 in a cohort of patients after MI. Interest in GDF3 as a marker of adverse cardiac remodeling arose from our preclinical studies in a mouse model of MI. Within 7 days of MI, a 7-fold increase in GDF3 mRNA expression and approximately a 2-fold increase in circulating GDF3 levels were observed in mouse hearts. These results were replicated in clinical samples derived from patients with MI. Thus, our results confirm a transient increase in GDF3 levels in a mouse MI model and suggest that cardiac PW1 plays a role in the regulatory process of scar tissue and cardiac fibroblast properties after MI. + The novel and important role of this marker as a paracrine factor secreted by cells is highlighted. Therefore, circulating GDF3 levels may be considered when evaluating the risk of adverse outcomes in patients after MI.

[0079] In a previous report, the inventors of this invention discussed activated cardiac PW1 + Pharmacological blockade of αV-integrin against cells led to in vitro activation of TGF-β and in vivo reduction of cardiac fibrosis after MI. 7 This observation and the involvement of GDF3 in TGF-β signaling. 11 This suggests a contribution of GDF3 to adverse cardiac remodeling after MI. We hypothesize the involvement of GDF3 in the inflammatory cascade during and after MI and support the concept of early intervention of GDF3 function in the inflammatory cascade to prevent myocardial damage. Risk stratification in the early stages after MI is challenging but useful for creating individualized treatment regimens in the future. Therefore, our study, supported by the correlation between circulating GDF3 levels, the post-MI scarring process, and cardiac function that we found, lays a strong foundation for future research seeking to target GDF3 in the treatment of MI.

[0080] Our clinical study focused specifically on post-MI patients who underwent cardiac MRI evaluation for cardiac remodeling, and is limited by a small sample size because this is not a routine study in these patients. Our results, based on imaging surrogates, indicate that patients with high GDF3 levels develop adverse cardiac remodeling, but further research is needed to validate its predictions regarding heart failure and cardiovascular outcomes. However, the majority of patients with high GDF3 levels had a significant decrease in LVEF (less than 50%) 6 months after MI. Furthermore, while we focused solely on the potential implications of GDF3, the roles of other upregulated markers, specifically PROK2 and NDP, were not investigated and require further research. In particular, the potential synergistic effects between these different secretory factors cannot be ruled out. Finally, this study investigated the role of secretory factors in fibroblast proliferation as a major underlying mechanism of cardiac fibrosis. However, other mechanisms include myofibroblast transformation. 27 or immune-inflammatory response28 This supports fibrotic alteration in ischemic hearts. The effects of GDF3 on these mechanisms warrant further investigation.

[0081] Conclusion: This specification shows that upregulation of the secreted protein GDF3 is detected in mouse and human plasma after MI. The levels of circulating GDF3 correlate with localized cardiac production in response to MI, and higher circulating levels of GDF3 are interpreted as indicating increased fibroblast proliferation and increased fibrosis. Consistent with this, we show that high levels of plasma GDF3 in a cohort of patients 4 days post-MI are consistent with poor outcomes measured at 6 months, including limited recovery of cardiac diastole and systolic function, and a higher number of akinetic segments. These data suggest that higher levels of circulating GDF3 can be used to identify patients who will develop adverse cardiac remodeling.

[0082] In conclusion, PW1 originating from ischemic heart + Cells release growth-promoting factors that induce the proliferation of resident fibroblasts. One such factor, GDF3, may serve as a novel marker for adverse fibrotic remodeling in cardiac tissue after MI. Its clinical applicability could enable the identification of patients at increased risk of severe cardiomyopathy and HF, as well as better and more specific disease management. table: [Table 1] [Table 2] TIFF2026102583000004.tif228165 TIFF2026102583000005.tif32165

[0083] Statistical analysis was performed using the Mann-Whitney nonparametric t-test for continuous variables and the chi-squared test (Fisher test for n<5) for bivariate variables. P<0.05 was obtained.

[0084] References: Throughout this application, various references disclose the state of the art relating to the present invention. The disclosures of these references are incorporated into this disclosure by reference. [Table 3] TIFF2026102583000007.tif230161 TIFF2026102583000008.tif129161

Claims

1. A method for determining whether a patient who has experienced a myocardial infarction has or is at risk of having adverse post-ischemic cardiac remodeling, comprising determining the level of GDF3 in a sample obtained from the patient, wherein the level indicates whether the subject has or is at risk of having adverse post-ischemic cardiac remodeling.

2. The method according to claim 1, wherein the sample is a blood sample, more specifically a serum sample.

3. A method according to claim 1, wherein the level of GDF3 is determined 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after myocardial infarction.

4. The method of claim 1, comprising contacting the sample with an agent that selectively binds to the GDF3 protein, particularly to the maturation domain of the protein.

5. The method according to claim 4, wherein the agent is an antibody.

6. A method according to claim 1, wherein the level of GDF3 is determined by an enzyme-linked immunosorbent assay.

7. A method according to claim 1, wherein a high level of GDF3 indicates a high probability that the patient will have or be at risk of having harmful post-ischemic cardiac remodeling, and conversely, a low level of GDF3 indicates a low probability that the patient will have or be at risk of having harmful post-ischemic cardiac remodeling.

8. below: The method of claim 1, comprising the steps of: i) quantifying the level of GDF3 in a sample obtained from a patient; ii) comparing the level quantified in step i) with a predetermined reference value; iii) concluding that if the level quantified in step i) is higher than the predetermined reference value, the patient has or is at risk of having harmful post-ischemic cardiac remodeling, or conversely, concluding that if the content quantified in step i) is lower than the predetermined reference value, the patient does not have or is not at risk of having harmful post-ischemic cardiac remodeling.

9. A method for treating adverse post-ischemic cardiac remodeling in patients who have experienced myocardial infarction, comprising administering a therapeutically effective amount of a GDF3 inhibitor to the subject.

10. The method according to claim 9, wherein the GDF3 inhibitor is an anti-GDF3 neutralizing antibody.

11. A method according to claim 10, wherein the anti-GDF3 neutralizing antibody binds to the mature domain of GDF3.

12. The method according to claim 11, wherein the anti-GDF3 neutralizing antibody binds to an amino acid sequence in the range from the amino acid residue at position 251 to the amino acid residue at position 364 in SEQ ID NO: 1.

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