GDF3 as a biomarker and biotarget in postischemic cardiac remodeling

Abstract

A robust marker of the scarring process in the early stage after myocardial infarction (MI) has not yet been identified, and it remains difficult to identify patients at high risk for developing the majority of adverse fibrotic remodeling and heart failure. Here, we demonstrate that resident PW1 in scarred cardiac tissue after MI. + We demonstrate the regulation of cellular paracrine behavior and the differential abundance of 12 candidate markers in their secretomes. Among these, growth differentiation factor 3 (GDF3), a member of the transforming growth factor-β family, upregulates cardiac fibroblast proliferation, which is instrumental to fibrosis. GDF3 is upregulated in scar tissue and plasma in mice and humans after myocardial infarction, with the highest plasma levels predicting greater fibrotic cardiac remodeling and cardiac dilation. Thus, we uncover a previously unidentified function of GDF3 in predicting adverse fibrotic cardiac remodeling after MI. Thus, the present invention relates to the use of GDF3 as a biomarker and biotarget in post-ischemic cardiac remodeling.

Description

[Technical Field]

[0001] The present invention is in the field of medicine, particularly cardiology. [Background technology]

[0002] Acute myocardial infarction (MI) is characterized by the death of 1 billion cardiomyocytes, or cardiac fibrosis, which activates adaptive repair processes and ultimately replaces the dead myocardium with a collagen-based scar. 1 A major goal of modern cardiovascular research is to reduce myocardial scarring, an important clinical predictor of mortality and sudden cardiac death. 2 Necrosis (increased cardiac troponin) 3 and cardiac dysfunction (increased brain natriuretic peptide) 4 While markers indicative of the scarring process have been identified, markers of the robust scarring process in the early stages after myocardial infarction have yet to be identified, and identifying patients at high risk for developing the majority of adverse fibrotic 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 the plasma membrane activin receptor-like kinase type 1 receptor B (ACVR1B, ALK4) and ACVR1C (ALK7) (Andersson, O.; Korach-Andre, M.; Reissmann, E.; Ibanez, C.F.; Bertolino, P. Growth differentiation 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 mice with endotoxin shock has been shown to increase survival 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 postischemic cardiac remodeling 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 post-ischemic cardiac remodeling.

[0005] Detailed description of the invention: Myocardial scar regression after acute myocardial infarction (MI) is a major cardiovascular research goal, but an incomplete understanding of the various sources of cardiac fibrosis poses a major challenge. This incomplete understanding has limited the identification of patient subgroups at higher risk for developing adverse fibrotic remodeling and heart failure. Here, we demonstrate the role of resident PW1 in scarred cardiac tissue after MI. + We demonstrate the regulation of cellular paracrine behavior and the differential abundance of 12 candidate markers in their secretomes. Among these, growth differentiation factor 3 (GDF3), a member of the transforming growth factor-β family, upregulates cardiac fibroblast proliferation, which is instrumental in fibrosis. GDF3 is upregulated in scar tissue and plasma in mice and humans after myocardial infarction, with its highest plasma levels predicting greater fibrotic cardiac remodeling and cardiac dilation. Thus, we uncover a previously unidentified function of GDF3 in predicting adverse fibrotic cardiac remodeling after MI.

[0006] Diagnostic Method: A first object of the present invention relates to 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 said level indicates whether the subject has or is at risk of having adverse post-ischemic cardiac remodeling.

[0007] As used herein, the terms "subject," "individual," or "patient" are used interchangeably and refer to any subject, particularly humans, 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 its common meaning in the art and relates to irreversible necrosis of the myocardium as a result of prolonged ischemia due to coronary thrombosis, i.e., the development of a thrombus in the major blood vessels that serve the heart.

[0009] As used herein, the term "detrimental post-ischemic cardiac remodeling" has its common meaning in the art and refers to significant changes that occur after myocardial infarction and can be detrimental to cardiac function. Cardiac remodeling includes molecular, cellular, and interstitial changes that clinically manifest as changes in cardiac size, shape, and function after myocardial infarction. For example, ventricular remodeling includes progressive ventricular hypertrophy accompanied by a decline in ventricular function. Myocyte function in cardiac muscle distant from the initially infarcted myocardium declines. Specifically, detrimental post-ischemic cardiac remodeling includes arrhythmias, cardiac dilation (assessed by left ventricular end-diastolic volume indexed by body surface area or LVEDV), and cardiac dysfunction (left ventricular ejection fraction or EF). Typically, detrimental post-ischemic cardiac remodeling is defined as a greater than 20% increase in left ventricular end-diastolic volume (LVEDV) at 6 months compared to the initial assessment (see Examples).

[0010] In some embodiments, the methods of the present invention are particularly suitable for determining whether a patient is at risk of having heart failure following a myocardial infarction.

[0011] As used herein, the terms "heart failure" or "HF" have their common meaning in the art and include congestive heart failure and / or chronic heart failure. 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 graded into four classes (I-IV). The classes (I-IV) are as follows: Class I: No limitation in any activity; no symptoms with usual activity; Class II: Mild activity limitation; patient is comfortable at rest or with mild exertion; Class III: Severe activity limitation; 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 specific period of time and can refer to the "absolute" or "relative" risk of a subject. Absolute risk can be measured either by referring to actual observational measurements for a relevant time cohort or by referring to an index value developed from a statistically valid historical cohort followed over a relevant period of time. Relative risk refers to the ratio of a subject's absolute risk to either the absolute risk of a low-risk population or the average population risk, and may vary depending on the method used to evaluate clinical risk factors. The odds ratio, which is the ratio of positive events to negative events for a given test result (odds are calculated by the formula p / (lp), where p is the probability of the event and (l-p) is the probability that the event will not occur), is also commonly used for non-conversion rates. "Risk assessment" or "assessing risk" in the context of the present invention encompasses predicting the probability, odds, or likelihood that an event or condition will occur, the likelihood that an event or condition will occur, or the incidence of an event or the rate of conversion from one condition to another. Risk assessment can also include prediction of future clinical parameters, values ​​of traditional laboratory risk factors, or other indicators of recurrence, either absolute or relative to a previously measured population. The methods of the present invention may be used to provide continuous or categorical measurements of conversion risk, i.e., to diagnose and define categorical risk regions for subjects defined as at risk of conversion. In categorical scenarios, the present invention can be used to distinguish between normal and other higher-risk subject populations. In some embodiments, the present invention can be used to distinguish at-risk populations from normal populations.

[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 methods according to the invention is a blood sample (e.g., a whole blood sample or a serum sample).

[0014] As used herein, the term "blood sample" refers to any blood sample derived from a subject. Collection of blood samples can be performed by methods well known to those of skill in the art. In some embodiments, the blood sample is a serum sample or a plasma sample.

[0015] In some embodiments, the level of GDF3 is 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 common meaning in the art and refers to growth / differentiation factor 3. An exemplary amino acid sequence of GDF3 is set forth as SEQ ID NO: 1. Typically, the term "GDF3" is understood by the presence of a mature domain ranging from the amino acid residue at position 251 to the amino acid residue at position 364 of SEQ ID NO: 1. [ka]

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

[0018] In some embodiments, the method comprises assessing protein levels in the sample by contacting the sample with an agent that selectively binds to a mature domain (such as an antibody or antigen-binding portion thereof), particularly as defined above. In some embodiments, the antibody bears a detectable label. The antibody can be polyclonal, or more preferably, monoclonal. An intact antibody, or an antigen-binding fragment 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 (i.e., physically linking) a detectable substance to the antibody, as well as indirectly labeling the antibody through 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, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, aequorin, and examples of suitable radioactive materials include 125 I, 131 I, 35 S or 3 Contains H.

[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, W.H. 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 the present methods for detecting and quantifying GDF3 protein in a sample. Such devices have been successfully used for 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 approaches can be used to isolate GDF3 protein from heterogeneous samples. For example, some techniques use functionalized surfaces that bind to target cell populations and can capture GDF3 using functionalized materials. Functionalized materials can include surface-bound capture moieties, such as antibodies, or other specific binding molecules, such as aptamers, as known in the art. Accordingly, such microfluidic chip technologies can be used in diagnostic and prognostic devices for use in the methods described herein. See, e.g., 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 microfluidic devices comprising a GDF3 binding moiety, e.g., an anti-GDF3 antibody or antigen-binding fragment thereof.

[0021] Typically, a high level of GDF3 indicates that a patient has a high probability of having or being at risk of having adverse post-ischemic cardiac remodeling, and conversely, a low level of GDF3 indicates that a patient has a low probability of having or being at risk of having adverse post-ischemic cardiac remodeling.

[0022] As used herein, the term "high" refers to an amount that is higher than normal, higher than a standard, such as the amount of a predetermined standard or subgroup, or relatively higher than the amount of another subgroup. For example, high GDF3 refers to an amount of GDF3 that is higher than the amount of normal GDF3. The amount of normal GDF3 may be determined according to any method available to those skilled in the art. High GDF3 may also refer to an amount that is equal to or higher than a predetermined standard, such as a predetermined cutoff. High GDF3 may also refer to an amount of GDF3, where the high GDF3 subgroup has a relatively higher level of GDF3 than another subgroup. For example, according to the present specification, two different patient subgroups can be created by dividing the sample around a mathematically determined point (such as, but not limited to, the median), thereby creating a subgroup with a high amount (i.e., higher than the median) and another subgroup with a low amount. In some cases, a "high" level may include a range of very high levels and a range of "moderately high" levels, where moderately high is a level higher than normal but lower than "very high."

[0023] As used herein, the term "low" refers to an amount that is lower than normal, lower than a standard, such as the amount of a predetermined standard or subgroup, or relatively lower than the amount of another subgroup. For example, low GDF3 refers to an amount of GDF3 that is lower than the normal amount of GDF3 in a particular series of patient samples. The normal amount of GDF3 may be determined according to any method available to those skilled in the art. Low GDF3 may also refer to an amount that is lower than a predetermined standard value, such as a predetermined cutoff. Low GDF3 may also refer to an amount in a low GDF3 subgroup that is relatively lower than another subgroup. For example, according to the present specification, two different patient subgroups can be created by dividing the samples around a mathematically determined point (such as, but not limited to, the median), thereby creating a group with a low amount (i.e., less than the median) compared to another group with a higher amount (i.e., more than the median).

[0024] In some embodiments, the method of the present invention comprises 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 the patient has or is at risk of having adverse post-ischemic cardiac remodeling if the level quantified in step i) is higher than the predetermined reference value, or conversely, concluding that the patient does not have or is not at risk of having adverse post-ischemic cardiac remodeling if the content quantified in step i) is lower than the predetermined reference value.

[0025] In some embodiments, the predetermined reference value is a threshold or cutoff value that can be determined experimentally, empirically, or theoretically. The threshold value 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, retrospective measurements of appropriately archived historical samples from subjects can be used to set the predetermined reference value. The threshold value must be determined to achieve optimal sensitivity and specificity based on test performance and benefit / risk balance (false-positive and false-negative clinical outcomes). Typically, optimal sensitivity and specificity values ​​(e.g., threshold values) can be determined using receiver operating characteristic (ROC) curves based on experimental data. For example, after quantifying the GDF3 level in a sample, statistical processing of the GDF3 levels measured in the tested sample can be performed using algorithmic analysis to obtain classification criteria that have significance for sample classification. The official name of the ROC curve is the receiver operator characteristic curve, and it is also known as a receiver operating characteristic curve. Receiver operating characteristic curves are 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 minus the specific value). The ROC curve uses image synthesis to reveal the relationship between sensitivity and specific value. A series of different cutoff values ​​(thresholds or critical values, the boundary between normal and abnormal results of a diagnostic test) are set as continuous variables, and a series of sensitivities and specific values ​​are calculated. A curve is then drawn using sensitivity as the ordinate and specific value as the abscissa. The larger the area under the curve (AUC), the higher the diagnostic accuracy. On an ROC curve, the point closest to the upper left corner of the plot is the critical point, which has both high sensitivity and high specific value. The AUC value of an ROC curve is between 1.0 and 0.5. When the AUC is greater than 0.5, the diagnostic result becomes increasingly better 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 greater than 0.9 indicates high accuracy. The algorithmic method is preferably computer-based.For drawing the ROC curve, existing software or systems in the art may be used, such as MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VI0.0 (Dynamic Microsystems, Inc. Silver Spring, Md., USA).

[0026] The method of the present invention is particularly suitable for identifying patients who may require additional attention and support after a 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 blockers, angiotensin-converting enzyme inhibitors, aldosterone antagonists, diuretics, hydralazine / nitrates, antithrombotic agents, beta-adrenergic receptor blockers, alpha-adrenergic receptor blockers, calcium channel blockers, etc. Examples of ACE inhibitors include, but are not limited to, captopril, benazepril, enalapril, lisinopril, fosinopril, ramipril, perindopril, quinapril, moexipril, and trandolapril. Examples of ARBs include losartan, candesartan, irbesartan, and valsartan. Examples of suitable β-blockers include, but are not limited to, alprenolol, carteolol, levobunolol, mepindolol, metipranolol, nadolol, oxyprenolol, penbutolol, pindolol, propranolol, sotalol, timolol, acebutolol, atenolol, betaxolol, bisoprolol, esmolol, metoprolol, nebivolol, carvedilol, celiprolol, labetalol, and butaxamine. Examples of diuretics include, but are not limited to, calcium chloride, ammonium chloride, amphotericin B, lithium citrate, goldenrod, juniper, dopamine, acetazolamide, dorzolamide, bumetanide, ethacrynic acid, 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 a myocardial infarction, comprising administering to the subject a therapeutically effective amount of a GDF3 inhibitor.

[0028] As used herein, the term "treatment" or "treating" refers to both preventative or prophylactic treatment and curative or disease-modifying treatment, including treatment of patients at risk of or suspected of having a disease, as well as patients who are ill or diagnosed as suffering from a disease or condition, and includes the suppression of clinical recurrence. Treatment is administered to a subject with a medical disorder or who is likely to eventually acquire the disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disease or recurrent disease, or to extend the subject's expected survival in the absence of such treatment. "Therapeutic regimen" refers to a pattern of disease treatment, e.g., the pattern of dosages used during treatment. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used in the initial treatment of a disease. The general purpose of an induction regimen is to provide high levels of drug to the patient during the initial period of the treatment regimen. The induction regimen may (partially or entirely) employ a "loading regimen," which involves administering a higher dose of drug than the physician uses during the maintenance regimen, administering the drug more frequently than the physician administers the drug during the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a patient during disease treatment, for example, to keep the patient in remission for an extended period of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, such as weekly, monthly, yearly, etc.) or intermittent therapy (e.g., discontinuing treatment, intermittent treatment, treatment upon relapse, or treatment upon the achievement of specific predetermined criteria (e.g., disease manifestation, etc.)).

[0029] Specifically, the method of the present invention is suitable for preventing or reducing damage to 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 reducing post-ischemic left ventricular remodeling. More specifically, the method of the present invention is suitable for increasing left ventricular ejection fraction (LVEF), and / or suppressing left ventricular dilation, and / or reducing 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 in the art or that will be identified in the future, including any chemical entity that, when administered to a patient, results in the 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 the amino acid sequence ranging from the amino acid residue at position 251 to the amino acid residue at position 364 of SEQ ID NO:1.

[0032] As used herein, the term "antibody" is therefore used to refer to any antibody-like molecule having an antigen-binding region, and 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, specifically incorporated herein by reference). Specifically, diabetes is further described in European Patent No. 404,097 and International Publication No. WO 93 / 11161, while linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating antibodies with pepsin. The resulting F(ab')2 fragments can be treated to reduce disulfide bridges to generate Fab' fragments. Papain digestion 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 techniques or chemically. Techniques for producing 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; Reiter et al., 1996, which further describes and allows for the production of effective antibody fragments.

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

[0034] In some embodiments, the antibodies of the invention are single domain antibodies. As used herein, the term "single domain antibody" has its general meaning in the art and refers to a single heavy chain variable domain of an antibody of the type found in camelid mammals, which do not naturally have light chains. Such single domain antibodies are also "nanobodies."

[0035] In some embodiments, the antibodies of the invention are fully human antibodies. As used herein, the term "fully human" refers to an immunoglobulin, e.g., an antibody or antibody fragment, where the molecule is entirely of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin. Fully human monoclonal antibodies can also be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Patent Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and the references cited therein, the contents of which are incorporated herein by reference.

[0036] In some embodiments, the antibodies of the invention are humanized antibodies. As used herein, "humanized" describes an antibody in which some, most, or all of the amino acids outside the CDR regions have been replaced with corresponding amino acids derived from a human immunoglobulin molecule. Methods of humanization include, but are not limited to, those described in U.S. Patent Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762, and 5,859,205, which are incorporated herein by reference.

[0037] In some embodiments, the GDF3 inhibitor is an aptamer. Aptamers are a class of molecules that represent an alternative to antibodies in terms of molecular recognition. Aptamers are oligonucleotide sequences capable of recognizing virtually any class of target molecule with high affinity and specificity. Such ligands may be isolated through systematic evolution of ligands by exponential enrichment of random sequence libraries (SELEX). Random sequence libraries can be obtained by combinatorial chemical synthesis of DNA, in which each member is ultimately a chemically modified linear oligomer of a specific sequence. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by platform proteins such as Escherichia coli thioredoxin A, selected from combinatorial libraries by the two-hybrid method (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 that has the biological effect of inhibiting gene expression. In a preferred embodiment of the present invention, the gene expression inhibitor is an siRNA, an antisense oligonucleotide, or a ribozyme. For example, antisense oligonucleotides, including antisense RNA molecules 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, thereby reducing the level, e.g., activity, of GDF3 in cells. For example, antisense oligonucleotides of at least about 15 bases and complementary to specific regions of the mRNA transcript sequence encoding GDF3 can be synthesized, for example, by conventional phosphodiester technology. Methods for using antisense technology to specifically inhibit gene expression of genes whose sequences are known are well known in the art (see, e.g., U.S. Patent Nos. 6,566,135, 6,566,131, 6,365,354, 6,410,323, 6,107,091, 6,046,321, and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression 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 causes the production of small double-stranded RNA, resulting in specific inhibition of GDF3 gene expression (i.e., RNA interference or RNAi). The antisense oligonucleotide, siRNA, shRNA, or ribozyme nucleic acid of the present invention can be delivered in vivo alone or in association with a vector. In the broadest sense, a "vector" is any vehicle capable of facilitating the transfer of an antisense oligonucleotide, siRNA, shRNA, or ribozyme nucleic acid into a cell, typically a cell that expresses GDF3. Typically, a vector transports the nucleic acid into a cell with less degradation than would occur in the absence of the vector.In general, vectors useful in the present invention include, but are not limited to, plasmids, phagemids, viruses, and other vehicles derived from viral or bacterial sources engineered by the insertion or incorporation of antisense oligonucleotides, siRNA, shRNA, or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector, and include, but are not limited to, nucleic acid sequences derived from the following viruses: Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; retroviruses such as adenovirus, adeno-associated virus, SV40 virus, polyomavirus, Epstein-Barr virus, papillomavirus, herpesvirus, vaccinia virus, poliovirus, and RNA viruses such as retroviruses. Other unnamed vectors known in the art 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, such as deoxyribonuclease I, cleave DNA relatively nonspecifically (regardless of sequence), while many, typically referred to as restriction endonucleases or restriction enzymes, cleave only at highly specific nucleotide sequences. The mechanism behind endonuclease-based genome inactivation generally requires a first step of DNA single- or double-strand breaks, which can then trigger two different cellular mechanisms for DNA repair that can be utilized for DNA inactivation: 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 its common meaning in the art and refers to clustered regularly interspaced short repeats associated with segments of prokaryotic DNA containing short repeats of a base sequence. In one embodiment, the endonuclease is CRISPR-cas9 from Streptococcus pyogenes. The CRISPR / Cas9 system is described in US Patent No. 8697359B1 and US Patent No. 2014 / 0068797.In one embodiment, the endonuclease is CRISPR-Cpf1, a more recently characterized CRISPR from Provotella and Francisella 1 (Cpf1) in 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 with 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 center of the chest; chest pain radiating to the jaw, teeth, shoulders, arms, and / or back; difficulty breathing or shortness of breath; epigastric discomfort with or without nausea or vomiting; and diaphoresis or sweating.

[0040] In some embodiments, the GDF3 inhibitor is administered simultaneously with or sequentially (i.e., before and after) a revascularization procedure performed on a subject. In some embodiments, the subject is administered the GDF3 inhibitor before, during, and after the revascularization procedure. In some embodiments, the subject is administered the GDF3 inhibitor as a bolus dose immediately before the revascularization procedure. In some embodiments, the subject is administered the GDF3 inhibitor continuously during and after the revascularization procedure. In some embodiments, the subject is administered the GDF3 inhibitor for a period 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; or 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; insertion of a bypass graft; insertion of a stent; directional coronary atherectomy; treatment with one or more thrombolytic agents; and removal of an obstruction.

[0041] A "therapeutically effective amount" refers to a sufficient amount of active ingredient to treat or alleviate symptoms at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope 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 specific compound employed; the specific composition employed; the subject's age, weight, general health, sex, and diet; the time of administration, route of administration, and excretion rate of the specific compound employed; the duration of treatment; drugs used in combination with the active ingredient; and similar factors well known in the medical arts. For example, it is well within the skill of the art to start administering the compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the product may vary over a wide range, from 0.01 mg to 1,000 mg per adult per day. Typically, the compositions contain 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 dosage to the treated subject. The pharmaceutical preparation typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. Usually, an effective amount of the drug is supplied at a dosage level of from 0.0002 mg to about 20 mg of body weight per day, particularly from 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 term "pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to mammals, particularly humans, as appropriate. 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 can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. 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 an isotonic agent, such as sugar or sodium chloride. Prolonged absorption of injectable compositions can be achieved by the use of absorption delaying agents, such as aluminum monostearate and gelatin, in the compositions. In the pharmaceutical compositions of the present invention, the active ingredient of the present invention can be administered in unit dosage form in admixture with a conventional pharmaceutical carrier. Suitable unit dosage forms include oral route forms such as tablets, gel capsules, powders, granules, and oral suspensions or solutions; sublingual and buccal dosage forms; aerosols; implants; subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, intrathecal, and intranasal dosage forms; and rectal dosage forms. [Brief explanation of the drawings]

[0043] The present invention is further illustrated by the following figures and examples, which should not be construed in any way as limiting the scope of the 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 non-failing (NF) (n=6) and failing (HF) hearts (n=9) from patients. *P<0.05 as determined by the 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 in non-remodelers (n=24) and remodelers (n=56) at 4 days after myocardial infarction. *P=0.05 analyzed by Mann-Whitney nonparametric t-test. [Figure 1c] GDF3 is a circulating factor secreted after myocardial infarction and can predict adverse cardiac remodeling in humans. c. ROC curve for discrimination between remodelers and non-remodelers. [Figure 1d] GDF3 is a circulating factor secreted after myocardial infarction and can predict adverse cardiac remodeling in humans. d–g. LVEDVi (d), LVEF (e), infarct size (f), and number of akinetic segments (g) 6 months after MI in patients from the low GDF3 group (<1375 pg / mL) and high GDF3 group (>1375 pg / mL). *P<0.05, **P<0.01 by Mann-Whitney nonparametric t-test. [Figure 1e] GDF3 is a circulating factor secreted after myocardial infarction and can predict adverse cardiac remodeling in humans. d–g. LVEDVi (d), LVEF (e), infarct size (f), and number of akinetic segments (g) 6 months after MI in patients from the low GDF3 group (<1375 pg / mL) and high GDF3 group (>1375 pg / mL). *P<0.05, **P<0.01 by Mann-Whitney nonparametric t-test. [Figure 1f]GDF3 is a circulating factor secreted after myocardial infarction and can predict adverse cardiac remodeling in humans. d–g. LVEDVi (d), LVEF (e), infarct size (f), and number of akinetic segments (g) 6 months after MI in patients from the low GDF3 group (<1375 pg / mL) and high GDF3 group (>1375 pg / mL). *P<0.05, **P<0.01 by Mann-Whitney nonparametric t-test. [Figure 1g] GDF3 is a circulating factor secreted after myocardial infarction and can predict adverse cardiac remodeling in humans. d–g. LVEDVi (d), LVEF (e), infarct size (f), and number of akinetic segments (g) 6 months after MI in patients from the low GDF3 group (<1375 pg / mL) and high GDF3 group (>1375 pg / mL). *P<0.05, **P<0.01 by Mann-Whitney nonparametric t-test. [Example]

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

[0045] animal research design Eight- or 13-week-old male C57BL / 6J mice and the PW1 reporter (PW1 nLacZMice underwent left anterior descending artery (LAD) surgery and were anesthetized in an induction chamber containing 2% isoflurane mixed with 100% O2 at 1.0 L per minute. The mice were placed in a supine position on a heating pad to maintain body temperature. The mice were intubated with an endotracheal tube and then connected to a rodent ventilator (180 breaths per minute, 200 μL tidal volume). Anesthesia was maintained with 1.5–2% isoflurane O2 throughout the surgical procedure. The chest was accessed from the left side through the intercostal space, and the pericardium was incised. The LAD was exposed and encircled proximally with an 8.0 prolene suture. The suture was briefly snare-ed, and the arterial area was blanched to confirm ligation. Mice were analyzed 7 days after permanent LAD ligation. Blood samples were collected into heparin-coated Eppendorf tubes and immediately centrifuged at 200 × g for 15 min at 4 °C to separate plasma, which was then stored at -80 °C until analysis. Hearts were excised and immediately digested for FACS sorting or qPCR analysis.

[0046] Cell separation and fluorescence-activated cell sorting (FACS) PW1 + CD51 + Cardiac cell sorting was performed as previously described 29 Briefly, small cell suspensions were prepared from 8-week-old PW1 mice. nLacZ Whole hearts were prepared 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 Biosciences), BUV395-tagged anti-TER119 (1:50 dilution, BD Biosciences), and phycoerythrin-cyanin 7-tagged anti-CD45 (1:500 dilution, eBioscience). To detect β-gal reporter activity, cells were incubated with the fluorescent substrate 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C). 12 The cells were incubated with FDG for 1 hour at 37°C. The various populations were gated, analyzed, and sorted using a FACSAria II cytometer (BD Biosciences).

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

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

[0049] Discard the reads that did not pass the Illumina filter and run them through the Cutadapt program. 30 After trimming low-quality sequenced bases (q<) using the q-seq command, downstream analysis was restricted to reads longer than 90 bp in length. Selected reads were then compared against the complete mouse reference genome and ENSEMBL. 32gtf transcript annotation file from the RSEM package 31 The transcripts were mapped to a mouse reference transcriptome generated by [translate]. The RSEM program was used to align and estimate transcript abundance for each of the 12 treated samples. Transcripts counted with an abundance 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, resulting in a profile of 16,403 genes. Analysis was performed in the R environment (version 3.2.2).

[0050] A Galaxy15.10 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). NLStradamus and PredictNLS were used in parallel to determine nuclear localization signals. Each dataset from RNA-seq corresponding to a different population was then processed through a pipeline designed to select sequences that did not contain signal peptides, transmembrane segments, or nuclear localization signals, as well as 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 from MI and SHAMC57BL / 6J mice 7 days after surgery using the RNAqueous Micro Kit (AM1931, Invitrogen) according to the manufacturer's instructions. 500 ng of extracted RNA was then subjected to reverse transcription using the SuperScriptIVVILO Kit (11756050, Invitrogen) according to 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 Scientific) under the following conditions: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds and 60°C for 1 minute, followed by 95°C for 10 seconds and 60°C for 1 minute. Expression of target genes was normalized to RPL13 expression and then analyzed using a -2% qPCR kit. ΔΔCT The analysis was carried out using the method.

[0052] HEK293 cell culture and transfection HEK293 cells were cultured in DMEM GlutaMax™-1 (Life Technologies) supplemented with 10% FBS and 1% penicillin and streptomycin at 37°C in the presence of 5% CO. HEK293 cells were plated at 6 x 10 cells per well in antibiotic-free medium. 5 Cells were plated on 1000-well plates. After 24 hours, expression plasmids (Origene and Genscript) were transfected using Lipofectamine™ 2000 (Life Technologies) according to the manufacturer's protocol, using 2 μg of plasmid and 6 μL of Lipofectamine™ 2000 diluted in Opti-MEM (Life Technologies). Cells were cultured for 2 days and then serum-starved for 8 hours. Conditioned media was then harvested and centrifuged at 200 × g for 10 minutes. The supernatant was stored at -80°C until MEF processing.

[0053] Isolation of mouse embryonic fibroblasts Primary MEFs were isolated from C57bL / 6J mouse embryos at 13.5 days post-coitum. Pregnant females were euthanized by cervical dislocation, and embryos were surgically removed and isolated from maternal tissues and yolk sacs in ice-cold phosphate-buffered saline (PBS). Embryos were then decapitated and eviscerated (heart, spleen, liver, and intestines were removed). The bodies were washed with ice-cold PBS to remove blood and then finely ground in a Petri dish without PBS. Samples were incubated in digestion solution (trypsin-EDTA solution 0.05% (Life Technologies), DNase 1 0.1 mg / mL (Sigma)) for 15 minutes at 37°C. The suspension was allowed to settle. The supernatant was poured off 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 the MEFs was resuspended in MEF medium. The pellet from the tissue digestion was resuspended in digestion solution and incubated at 37°C for 15 minutes. The cells were allowed to settle, the supernatant was poured off, and the cells were processed as described above. Cells from the first and second digestion stages were pooled and plated into Petri dishes. Each 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 passaged when they reached 80% confluence. MEFs were harvested by trypsinization, centrifuged, and resuspended in freezing medium (DMEM 4.5 g / L D-glucose, 1% penicillin-streptomycin, 10% dimethyl sulfoxide). Primary MEFs were cultured between passages 0 and 4.

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

[0056] As mentioned above 7After separation of cardiomyocytes and non-cardiomyocytes from adult mouse hearts, proteins were loaded onto NuPAGE™ Novex™ 4-12% Bis-Tris gels (Life Technologies) and denatured at 70°C for 10 minutes. After 3 hours of electrophoresis, proteins were transferred onto nitrocellulose membranes using a Trans-Blot Turbo Transfer System (Bio-Rad) and stained with 0.1% Ponceau S (w / v in 5% acetic acid) to assess transfer quality and uniform loading. The membranes were blocked for 1 hour in Tris-buffered saline containing 5% skim milk and 0.1% Tween-20 (TBS-Tween) with constant shaking and then incubated overnight at 4°C with primary antibodies specific for 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 horseradish 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), after which they were imaged with a Chemidoc™ XRS+ camera (Bio-Rad) and analyzed using ImageLab™ software.

[0057] ELISA GDF3 levels were measured using a GDF3 sandwich ELISA assay (GenWay, GWB-KBBHW6) according to the manufacturer's instructions. Briefly, standards and diluted samples (1:16 in standard diluent) were added to an anti-GDF3 microplate (pre-coated with a GDF3-specific antibody) and incubated for 1 hour at 37°C. After removal of the standards and samples, a biotinylated GDF3 detection antibody was applied. The plate was incubated for 1 hour at 37°C. The wells were washed and then incubated with an avidin-HRP conjugate for 30 minutes at 37°C. Finally, after thorough washing, the wells were incubated with 3,5,3',5'-tetramethylbenzidine (TMB) substrate for 15 minutes at 37°C in the dark. The blue product resulting from oxidation of the TMB substrate turned yellow after 15 minutes of incubation at 37°C, following termination of the reaction by the addition of 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] PREGICA cardiac MRI sub-study We used a plasma bank from 80 patients with first-time STEMI who enrolled in the prospective PREGICA cardiac MRI substudy (Predisposition Genetical in Cardiac Insufficiency, clinicaltrials.gov identifier NCT01113268). Details of this study have been previously described (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 French cardiology centers and enrolled patients aged 18 to 80 years who presented within 24 hours of symptom onset with a first STEMI referred between 2010 and 2017. STEMI was defined by the presence of ST-segment elevation on the ECG, a significant troponin elevation (≥3x upper reference limit), and the presence of at least three akinetic LV segments on the initial transthoracic echocardiogram. Patients were excluded if they had persistent atrial fibrillation, a previous diagnosis of MI, or a history of cardiac disease. All patients underwent coronary angiography and primary PCI within the first 24 hours. In a subset of patients defined in the CMR substudy of the PREGICA cohort, Cardiac MRI was performed using a 1.5T unit at 4 ± 2 days after admission and at 6-month follow-up. A standardized MRI protocol was followed at all centers, and images were centrally analyzed. Cine images were acquired in the long-axis and short-axis views using a breath-hold steady-state free precession sequence. Left ventricular (LV) volumes and ejection fraction (EF) were derived using a stack of short-axis slices covering the atrioventricular annulus to the apex. Ten minutes after intravenous injection of a gadolinium-based contrast agent, late gadolinium enhancement (LGE) images were acquired in the same long-axis and short-axis views as the cine images using a breath-hold segmented T1-weighted inversion recovery gradient echo sequence. LGE images were used to assess infarct size. Blood samples were collected simultaneously with cardiac MRI.GDF3 was quantified using an ELISA assay in available plasma collected on day 4 (n = 80). The study was approved by the institutional review board, and all patients provided written informed consent.

[0059] statistical analysis Mouse and in vitro studies. The number of samples (n) used in each experiment is recorded in the text and figure legends. All experiments were performed at least twice independently. Data are expressed as mean ± standard deviation (SD). Quantitative data were analyzed using one-way analysis of variance (ANOVA, Kruskal-Wallis test) and pairwise comparisons with 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 the PREGICA cardiac MRI substudy. P values ​​were obtained from the chi-squared test statistic for dichotomous variables 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 linear regression models with additional adjustment for age and sex.

[0061] All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA). A value of P < 0.05 indicated statistical significance.

[0062] result PW1 derived from ischemic heart + The cells release factors that promote the proliferation of fibroblasts. Adult PW1 nLacZ Reporter mice were transfected as previously described. 6,7 Heart injury was induced by ligation of the left anterior descending coronary artery (LAD). On day 7 after injury, hearts were harvested from MI mice, as well as from SHAM-operated mice, and analyzed by fluorescence-activated cell sorting (FACS). +The cells were isolated (not shown). After 5 days of culture, the conditioned medium was collected from these cells and used to culture mouse embryonic fibroblasts (MEFs) for 24 hours (not shown). The effect of the conditioned medium on the proliferation of MEFs was assessed using a CyQUANT™ cell proliferation assay. The results of the cell proliferation assay were compared between the conditioned medium from control cells or PW1 cells derived from SHAM-operated hearts. + PW1 isolated from ischemic hearts compared with those treated with + A significant increase in proliferation of MEFs incubated with conditioned medium from PW1 cells was demonstrated (not shown). - There was no corresponding significant increase in conditioned medium from cells (not shown). These observations suggest that activated PW1 cells from ischemic hearts + It suggests that the cells may release growth-promoting factors that induce proliferation of resident fibroblasts.

[0063] RNA sequencing (RNA-seq) and bioinformatics analysis predict potential biomarkers involved in paracrine effects. FACS-isolated PW1 from SHAM and MI mice + The transcriptome of PW1 cells was characterized by RNA-seq. + We investigated the effect of the ischemic cardiac environment on the paracrine potential of cells. RNA-seq output files were filtered, aligned, and quality-controlled to obtain a list of transcripts showing the greatest signal intensity (not shown). Cardiac PW1 + A comparative analysis was performed to understand disease-induced changes in the secretory behavior of final candidates with expression levels more than two-fold higher under ischemic conditions than under normal conditions (not shown). Subsequently, the predicted amino acid sequences of the corresponding genes were examined by a series of bioinformatics algorithms to identify secreted proteins. Proteins with a predicted N-terminal endoplasmic reticulum (ER)-targeting signal peptide but no predicted transmembrane domain or subcellular localization signal (i.e., no ER retention signal, mitochondrial targeting peptide, or nuclear export signal) were considered (not shown). Progressive filtering revealed that cardiac PW1 expression levels were significantly higher under ischemic conditions than those under normal conditions. +A total of 24 secreted proteins were overexpressed by the cells (not shown), and quantitative polymerase chain reaction (qPCR) confirmed that the expression of 12 of these 24 candidates was significantly increased in ischemic hearts (remote or infarcted regions) compared with normal hearts (not shown).

[0064] MI-activated PW1 compared with the secretome of control cells + The cellular secretome contained several growth factors, cytokines, and enzymes, as well as several less well-characterized factors (not shown). Secretion of growth factors GDF3, cytokines such as NDP and CCL8, and enzymes such as CELA1 and PRTN3 was more than two-fold higher in MI hearts than in SHAM hearts (not shown).

[0065] Transfection experiments will identify candidates that have a proliferative effect on fibroblasts. We then examined the effects of the candidates on the proliferation of cultured embryonic and adult cardiac fibroblasts. Six candidates (CCL8, CELA1, GDF3, NDP, PRNT3, and PROK2) were selected based on their gene ontology biological functions, which indicated they might be associated with cell proliferation. Conversely, we excluded lipoproteins APOC2, APOC4, and SAA3, coagulation factor F10, and the less well-characterized C1QTNF3 and DMKN. The cDNAs for these six proteins were separately cloned into mammalian expression plasmids, which were then used to transfect HEK-293 cells (not shown). FLAG epitope-tagged fibroblast growth factor 23 cDNA served as a positive control, while an empty vector served as a negative control. Forty-eight hours after transfection, conditioned medium was collected and tested to confirm overexpression of secreted proteins (not shown), and then used to incubate serum-starved MEFs. Assessment of cell proliferation rates after 24 hours of treatment revealed four factors that significantly induced MEF proliferation compared with control treatment: growth differentiation factor-3 (GDF3), Norrin cysteine ​​knot growth factor (NDP), prokineticin 2 (PROK2), and chymotrypsin-like elastase family member 1 (CELA1) (data not shown). The proliferative effects of three of these four candidates (GDF3, NDP, and PROK2) were further confirmed using freshly isolated adult cardiac fibroblasts (data not shown). Thus, cell proliferation assays facilitated the selection of three candidates, GDF3, NDP, and PROK2, from the 12 overexpressed markers.

[0066] Of these three remaining candidates, GDF3 (also known as Vg-related gene 2) was found to be the most predominantly overexpressed in the ischemic heart and to be one of the most significant increases in fibroblast proliferation. GDF3 is a member of the TGF-β superfamily, consisting of 366 amino acid residues. Human and mouse GDF3 share 76.6% nucleotide homology and 69.3% peptide identity. 12The predicted amino acid sequence contains a signal sequence for secretion at the hydrophobic NH2 terminus, a prodomain that promotes cysteine-mediated disulfide bond formation with other family members, and a putative proteolytic processing site at 114 amino acid residues (not shown). Cleavage of GDF3 at this residue generates the mature GDF3 protein, which is 114 residues long. 11 GDF3 plays an important role in early development in mice and humans. 13 , its expression is low in adult organs and particularly negligible in the adult heart 10、14、15 While the function and implications of GDF3 in the adult heart remain unclear, the biological function of GO suggests its involvement in the SMAD protein signaling pathway, which is highly relevant to the process of cardiac fibrosis. This is consistent with the strongest proliferative effect of GDF3 among all candidates on MEFs (not shown). Transfection experiments (not shown) confirmed that GDF3 is a secreted protein, as indicated by a full-length protein band on Western blots of supernatants (not shown). These observations, taken together, suggest a potential role for GDF3 in regulating fibroblast proliferation in scar tissue, prompting us to investigate the expression profile of GDF3 in mouse and human MI hearts.

[0067] GDF3 levels are increased in the plasma and infarct region of mouse hearts after MI. Based on the transcriptome results, we attempted to evaluate GDF3 expression in the whole heart by Western blotting and determine its cellular source. We detected the mature form of GDF3 in both neonatal and adult normal hearts (not shown). Specifically, in adult hearts, GDF3 was expressed only in the non-cardiomyocyte fraction, but not in cardiomyocytes (not shown). Further analysis of the non-cardiomyocyte fraction revealed that the specific expression of GDF3 was localized in the PW1 + confirmed in cells, but not in normal hearts. -However, this was not confirmed 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 hearts were excised 7 days later. GDF3 expression was analyzed separately in the infarcted and remote regions, which correspond to scar tissue. Western blotting confirmed that GDF3 expression in the infarcted region of MI hearts was higher than that in the corresponding region of SHAM hearts (not shown). This result is consistent with our previous observations and the finding that cardiac PW1 responds to MI. + Consistent with the fibrogenic fate of the cells, we demonstrate that GDF3 is produced at the infarct site, suggesting its involvement in the scarring process after MI.

[0068] GDF3 is a circulating factor that is secreted after MI. Given the secreted nature of this protein, we investigated whether free GDF3 could be detected in the circulation by analyzing plasma samples from MI and SHAM mice. Similarly, Western blot analysis confirmed that higher levels of mature GDF3 were present in the plasma of MI mice than in the plasma of SHAM mice (not shown). A GDF3-specific enzyme-linked immunosorbent assay (ELISA) was performed to examine the time course of circulating GDF3 levels in mouse plasma. Secreted protein levels increased from day 0 to day 2 after MI and then decreased until day 7.

[0069] Overall and cardiac PW1 + In line with the fibrogenic fate of the cells, these results indicate that GDF3 may be a novel myocardium-derived factor (cardiokine) secreted by these cells that may be involved in adverse cardiac remodeling after MI.

[0070] Circulating GDF3 levels serve as a marker of adverse remodeling after MI in humans. To examine the clinical relevance of our findings in a mouse MI model, we first assessed GDF3 expression in left ventricular cardiac tissue samples taken from failing, ischemic, and non-failing hearts of patients. Western blot analysis revealed stronger GDF3 expression in failing hearts than in non-failing hearts (Fig. 1a), indicating that upregulation of cardiac GDF3 expression levels 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 (Genetic Predisposition in Heart Failure [PREGICA] Patient Collection, NCT01113268) with a first-ever acute ST-segment elevation myocardial infarction (STEMI) who presented within 24 hours of symptom onset and were treated with primary percutaneous coronary intervention (PCI). Patients underwent initial clinical and biological evaluation on day 4 and serial cardiac magnetic resonance imaging (MRI) at 4 days and 6 months after angioplasty. Details of the inclusion / exclusion criteria are described in https: / / clinicaltrials.gov / ct2 / show / NCT01113268. The baseline characteristics of these patients are shown in Table 1.

[0072] First, left ventricular end-diastolic volume (LVEDV) indexed to body surface area (LVEDVi, mL / m) was measured at 6 months compared with the initial assessment with cMRI. 2Adverse cardiac remodeling was defined as a 20% or greater increase in GDF3 (pg / mL). Patients were accordingly classified as remodelers (n = 24) or non-remodelers (n = 56). GDF3 was detectable in the plasma of these patients measured on day 4 after PCI, and levels were significantly higher in remodelers than in non-remodelers (1364 ± 521 vs. 1090 ± 532 pg / mL, p = 0.033) (Fig. 1b). After adjusting for age and gender, a 1-standard deviation (SD) increase in GDF3 levels 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 statistical differences based on gender, smoking, history of hypertension, or diabetes (p > 0.10). Of note, GDF3 levels were moderately correlated with CRP (p = 0.13) and Hb1AC (p = 0.18), even though these correlations did not reach statistical significance (p = 0.28 and p = 0.12, respectively). To better evaluate the relationship between cardiac remodeling and plasma GDF3 levels, we first divided patients into four quartiles of GDF3 levels (measured on day 4 post-MI) and compared LVEDVi and LVEF measured by cardiac MRI at 6 months post-MI between these quartiles. We found that patients with the highest GDF3 levels (i.e., quartile 4) had significantly higher LVEDVi and lower LVEF compared with patients with lower GDFR3 levels. Next, we performed receiver operating characteristic (ROC) curve analysis to determine whether GDF3 levels could help distinguish the two groups. The area under the receiver operating characteristic curve (ROC) curve for the age- and sex-adjusted model was significantly different (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 (Fig. 1c).

[0073] Patients were then classified according to this cutoff value (≥1375 pg / mL, n = 25, referred to as high GDF3; <1375 pg / mL, n = 55, referred to as low GDF3). Table 2 reports the main characteristics and cardiac MRI findings at baseline and 6 months after MI in both groups. There were no significant imbalances in major cardiovascular risk factors between the two groups. Patients with high GDF3 levels (P < 0.05) had a significantly longer delay (up to 1.2 hours) between symptom onset and coronary artery occlusion. However, the peak of troponin, a surrogate marker of myocardial necrosis, was significantly lower in the high GDF3 group. Regarding cardiac remodeling, patients with high GDF3 showed no tendency to have significantly greater cardiac dilation than those in the low GDF3 group at 4 days after MI. However, in patients with high GDF3, LVEDVi values ​​at 6 months after MI were significantly higher (P<0.005) (Fig. 1d) and abnormal (normal value <82 mL / m 2 ), indicating adverse cardiac remodeling with the progression of cardiac dilatation. These patients also showed a significant decline in LVEF (p<0.05) at 4 days and 6 months, suggesting a reduced recovery of systolic function after MI in these patients (Figure 1e). While total infarct size (% of total heart weight) was not significantly different between the two groups (Figure 1f), patients with high GDF3 levels had a higher proportion of akinetic segments on cardiac MRI at 6 months compared with patients with low GDF3 levels (Figure 1g). This result indicates a more significant pathological transformation of the infarct area into a noncontractile scar in patients with high GDF3 levels, highlighting the diagnostic importance of GDF3 as a marker of adverse cardiac remodeling after MI.

[0074] Consideration: Acute myocardial infarction characterized by left ventricular remodeling may progress to the development of heart failure. 16 Markers reflecting myocardial damage may not 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 1). 17、18For example, galectin-3 was shown to be involved in fibrosis and inflammation and to be independently associated with the development of peripheral arterial disease in an observational study that included only white and black individuals and did not exclude the influence of confounding factors. Therefore, discovering potential markers that provide information on the preclinical stage of HF is essential for identifying patients at high risk for HF and providing timely disease management.

[0075] In an effort to contribute to wound healing, the cardiac ECM undergoes constant remodeling upon injury 19 Interestingly, the concept of ECM regulation via key molecules involved in cell-cell communication has only recently emerged. Herein, we investigate cardiac PW1, a cell subpopulation that is skeptical of orchestrating repair processes in tissues, including the heart. + cell 6、7 We focused on these PW1 + We investigated key differences in the secretome of interstitial cells. Notably, cardiac PW1 cells isolated from ischemic mouse hearts + The growth-promoting effect observed in conditioned medium from cardiac PW1 cells isolated from normal mouse hearts was similar to that observed in conditioned medium from cardiac PW1 cells isolated from normal mouse hearts. + Not only cells but also cardiac PW1 - RNA sequencing and bioinformatics analysis revealed that several factors in MI-activated cardiac PW1 + Upregulation of the expression of 12 secreted factors by cells, specifically GDF3, PROK2, and NDP, was confirmed, which was confirmed by qPCR validation experiments (not shown), showing approximately 7-fold, 3-fold, and 3-fold upregulation of expression after MI. Furthermore, the 12 candidate dysregulated markers confirmed by qPCR validation were mostly enriched in GO biological processes such as angiogenesis, inflammation, chemotaxis, and proliferation, thus suggesting a role for PW1 in MI. + Significant responses of cell populations were identified.

[0076] MI is characterized by an acute inflammatory response that contributes to 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 of cardiac fibrosis. 23、24 .

[0077] GDF3, a member of the TGF-β superfamily, was initially shown to be involved in early embryonic development, muscle development, adipose tissue homeostasis, and energy balance through interactions with the activin receptor-like kinase type 1 receptor B (ACVR1B, ALK4) and ACVR1C (ALK7) receptors. 25 Recent studies have highlighted the important role of GDF3 in macrophage function and the inflammatory cascade. Wang et al. recently described the role of GDF3 in macrophage polarization and endotoxin / sepsis-induced cardiac injury. 26 This study identifies a previously unrecognized function of GDF3 in cardiac fibrosis and demonstrates 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 prognostic potential of GDF3 in a cohort of post-MI patients. Interest in GDF3 as a marker of adverse cardiac remodeling arose from our preclinical studies in a mouse model of MI. We observed a 7-fold increase in GDF3 mRNA expression in mouse hearts and an approximately 2-fold increase in circulating GDF3 levels within 7 days of MI. These results were replicated in clinical samples from patients with MI. Thus, our results confirm the transient increase in GDF3 levels in a mouse MI model and suggest a role for cardiac PW1 in the process of regulating scar tissue and cardiac fibroblast properties after MI. + This highlights the novel and important role of this marker as a paracrine factor secreted by cells. Therefore, the levels of circulating GDF3 may be taken into consideration while assessing the risk of adverse outcomes in patients after MI.

[0079] In a previous report, the inventors reported that activated cardiac PW1 + Pharmacological blockade of αV-integrin on cells reduced TGF-β activation in vitro and cardiac fibrosis after MI in vivo. 7 This observation and the involvement of GDF3 in TGF-β signaling 11 These findings suggest the contribution of GDF3 to adverse cardiac remodeling after MI. We speculate on the involvement of GDF3 in the inflammatory cascade during / 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 stage after MI is challenging but may be useful in developing personalized treatment regimens in the future. Therefore, our study, supported by the association we found between circulating GDF3 levels, the post-MI scarring process, and cardiac function, lays a strong foundation for future research targeting GDF3 in the treatment of MI.

[0080] Our clinical study focused specifically on post-MI patients undergoing cardiac MRI evaluation for cardiac remodeling, a study not routinely performed in these patients, and is therefore limited by a small sample size. Our results, based on imaging surrogates, indicate that patients with high GDF3 levels develop adverse cardiac remodeling, but further studies will be needed to validate its prediction regarding heart failure and cardiovascular outcomes. However, the majority of patients with high GDF3 levels had significantly reduced LVEF (<50%) 6 months after MI. Furthermore, while we focused solely on the potential implications of GDF3, the role of other upregulated markers, specifically PROK2 and NDP, was not investigated and warrants further study. Notably, potential synergistic effects between these different secreted factors cannot be excluded. Finally, this study investigated the role of secreted factors on fibroblast proliferation as a major mechanism underlying cardiac fibrosis. However, other mechanisms, such as myofibroblast transformation, may also be involved. 27 and immune inflammatory responses28 These mechanisms support the fibrotic transformation of the ischemic heart. The influence of GDF3 on these mechanisms deserves further investigation.

[0081] Conclusion: This paper demonstrates that upregulation of the secreted protein GDF3 is detected in the plasma of mice and humans after MI. Circulating GDF3 levels correlate with local cardiac production in response to MI, with higher circulating GDF3 levels interpreted as indicating increased fibroblast proliferation and fibrogenesis. Consistent with this, we demonstrate that high plasma GDF3 levels in a cohort of patients 4 days after MI correlate with poor outcomes measured 6 months later, including cardiac dilation, limited recovery of systolic function, and a higher number of akinetic segments. These data suggest that higher circulating GDF3 levels can be used to identify patients who will develop adverse cardiac remodeling.

[0082] In conclusion, PW1 derived from ischemic heart + These cells release growth-promoting factors and induce the proliferation of resident fibroblasts. One such factor, GDF3, may serve as a novel marker of adverse fibrotic remodeling in cardiac tissue after MI. Its applicability in clinical settings may enable the identification of patients with severe myocardial fibrosis and increased risk of HF, as well as better and more specific disease management. table: [Table 1] [Table 2] TIFF0007823034000004.tif228165 TIFF0007823034000005.tif32165

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

[0084] References: Throughout this application, various references disclose the state of the art to which this invention pertains. The disclosures of these references are incorporated by reference into this disclosure. [Table 3] TIFF0007823034000007.tif230161 TIFF0007823034000008.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, the method comprising determining the level of GDF3 in a sample obtained from the patient, wherein the level indicates whether the patient has or is at risk of having adverse post-ischemic cardiac remodeling.

2. The method of claim 1 , wherein the sample is a blood sample.

3. The method of claim 2 , wherein the blood sample is a serum sample.

4. 2. The method of 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 the myocardial infarction.

5. 10. The method of claim 1, comprising contacting the sample with an agent that selectively binds to the GDF3 protein.

6. The method of claim 1, comprising contacting the sample with an agent that selectively binds to the mature domain of GDF3.

7. The method of claim 5 or 6, wherein the agent is an antibody.

8. 10. The method of claim 1, wherein the level of GDF3 is determined by enzyme-linked immunosorbent assay.

9. The method of claim 1, wherein a high level of GDF3 indicates a high probability that the patient has or is at risk of having adverse post-ischemic cardiac remodeling, and conversely, a low level of GDF3 indicates a low probability that the patient has or is at risk of having adverse post-ischemic cardiac remodeling.

10. i) quantifying the level of GDF3 in the sample obtained from the patient; ii) comparing the level quantified in step i) with a predetermined reference value; and iii) indicating that the patient has or is at risk of having adverse post-ischemic cardiac remodeling if the level quantified in step i) is higher than the predetermined reference value, or conversely indicating that the patient does not have or is not at risk of having adverse post-ischemic cardiac remodeling if the level quantified in step i) is lower than the predetermined reference value.

2. The method of claim 1, comprising:

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