WO2014064292A1

WO2014064292A1 - A method for preventing or treating atrial fibrillation

Info

Publication number: WO2014064292A1
Application number: PCT/EP2013/072521
Authority: WO
Inventors: Karine Clement; Elise Balse; Nicolas VENTECLEF; Stéphane HATEM
Original assignee: Universite Pierre Et Marie Curie (Paris 6)
Priority date: 2012-10-26
Filing date: 2013-10-28
Publication date: 2014-05-01

Abstract

The invention relates to the use of an activin inhibitor, such as an activin neutralizing antibody, in preventing or treating atrial fibrillation in a patient, especially a patient who shows epicardial fat.

Description

A method for preventing or treating atrial fibrillation

The present invention relates to the prevention or treatment of atrial fibrillation. Background of the invention:

As obesity and type 2 diabetes are becoming an epidemic in westernized countries, the incidence and prevalence of obesity- and diabetes-related heart diseases such as coronary artery disease, atrial fibrillation (AF), non-ischemic cardiomyopathies and heart failure are increasing.

Atrial fibrillation is the most common cardiac arrhythmia. It is often associated with palpitations, fainting, chest pain, or congestive heart failure. AF increases the risk of stroke; the degree of stroke risk can be up to seven times that of the average population, depending on the presence of additional risk factors (such as high blood pressure). It may be identified clinically when taking a pulse, and the presence of AF can be confirmed with an electrocardiogram (ECG) which demonstrates the absence of P waves together with an irregular ventricular rate.

In AF, the normal regular electrical impulses generated by the sinoatrial node are overwhelmed by disorganized electrical impulses usually originating in the roots of the pulmonary veins, leading to irregular conduction of impulses to the ventricles which generate the heartbeat. AF may occur in episodes lasting from minutes to days ("paroxysmal"), or be permanent in nature. Recently, the epicardial adipose tissue (EAT) emerges as an important actor of the pathogenesis of metabolic-related cardiac diseases. This ectopic fat deposit surrounding the myocardium and coronary arteries without any separation between the cardiomyocytes or adventitia is a metabolically active tissue which produces pro-atherogenic and proinflammatory adipokines (lacobellis et al, Nat Clin Pract Cardiovasc Med 2005;2(10):536-43; Mazurek et al, Circulation 2003;108(20):2460-6; Keophiphath et al, Mol Endocrinol 2009;23(1 ):1 1 -24; Ouwens et al, J Cell Mol Med 2010;14(9):2223-34). In other adipose tissue depots, it has been described a paracrine dialogs between adipocytes, preadipocytes, endothelial and inflammatory cells resulting in the release of a myriad of pro- and antiinflammatory molecules leading to a low-grade inflammatory and pro-fibrotic environment (Divoux et al, Diabetes 2010;59(1 1 ):2817-25; Dalmas et al, Trends Immunol 201 1 ;32(7):307- 14). The biological activity of EAT is related to the severity of coronary artery disease (CAD) (Ouwens et al, J Cell Mol Med 2010;14(9):2223-34; Eroglu et al, Nutr Metab Cardiovasc Dis 2009;19(3):21 1 -7; Dutour et al, J Clin Endocrinol Metab 2010;95(2):963-7) and the degree of cardiac hypertrophy (lacobellis et al, Am J Cardiol 2004;94(8):1084-7; Fox et al, Circulation 2009;1 19(12):1586-91 ). Recently it has been reported that the thickness of EAT is enhanced in patients with chronic AF or with atrial dilation (Al Chekakie et al, J Am Coll Cardiol 2010;56(10):784-8; Nagashima et al, Circ J 201 1 ;75(1 1 ):2559-65; Thanassoulis et al, Circ Arrhythm Electrophysiol 2010;3(4):345-50; Tsao et al, . Am J Cardiol 201 1 ;107(10):1498-503; Wong et al J Am Coll Cardiol 201 1 ;57(17):1745-51 ).

Treatment of atrial fibrillation (AF) generally aims at preventing temporary circulatory instability and stroke. Control of heart rate and rhythm are principally used to achieve the former, while anticoagulation may be required to decrease the risk of stroke. In emergencies, when circulatory collapse is imminent due to uncontrolled rapid heart rate, immediate cardioversion may be indicated.

However there remains a need for a treatment that would prevent atrial fibrillation to occur and establish.

Summary of the invention:

The inventors now propose to use activin inhibitors to prevent or treat atrial fibrillation.

A subject of the invention is thus an activin inhibitor, in particular an activin inhibitor substance, for use in preventing or treating atrial fibrillation in a patient, especially in a patient at risk of atrial fibrillation. Preferably, the patient shows epicardial fat, and in particular an excess of epicardial fat.

The activin inhibitor may be selected from the group consisting of an anti-activin antibody, follistatin, an inhibitor to activin receptor, an anti-activin receptor antibody and an anti-activin antisense DNA or iRNA. Preferably, the activin inhibitor is selected from the group consisting of an anti-activin antibody, follistatin and an inhibitor to activin receptor. The activin inhibitor may also be selected from the group consisting of an anti-activin antibody, follistatin and an anti-activin antisense DNA or iRNA.

In particular, the activin inhibitor may be an activin neutralizing antibody. In a preferred embodiment, the activin inhibitor inhibits activin A.

More particularly, the activin inhibitor may be an activin neutralizing antibody that binds and inhibits activin A, preferably that specifically binds and inhibits activin A.

Preferably, the activin inhibitor is to be administered intravenously.

In a particular embodiment, the atrial fibrillation to be treated is chronic.

The activin inhibitor may inhibit myocardial remodeling and more particularly fibrotic myocardial remodeling.

It is also described a method for preventing or treating atrial fibrillation in a patient, which method comprises administering an activin inhibitor in a patient in need thereof, especially in a patient who shows epicardial fat. In a preferred embodiment, the activin inhibitor inhibits activin A. Legends to the Figures:

Figure 1. Activin A expression increased in epicardial adipose tissue. (A and B) Activin A and Follistatin gene expression were measured in paired samples of epicardial and subcutaneous (SAT) adipose tissue from 12 subjects (Study population: Group 1 ). All results are normalized versus 18S gene expression levels. Ratios of Activin A / Follistatin have been measured at the mRNA and protein levels (C-D). (E) Activin A immuno-staining of paired samples of epicardial and subcutaneous (SAT) adipose tissue from 12 subjects (Study population: Group 1 ). (E) Quantification of Activin A staining by measuring the intensity of the signal (Pixels). The nonparametric Wilcoxon test was used. Data are represented by mean ± SEM. ^*P<0.001 .

Figure 2. Neutralization of Activin A prevents atrial fibrosis. Atria were co-incubated for one week with epicardial adipose tissue conditioned media or control media (Ctrl) pre-incubated with the Activin A-neutralizing monoclonal antibody or immune IgG. (A) Fibrosis was visualized using picrosirius red. Sarcomeric oc-actinin immuno-staining confirmed that samples were well conserved (right panel). (B) Total fibrosis: data depict the amount of fibrotic area as a percentage of the total tissue surface. ^*P<0.01 (C) Data are expressed in the percentage of fibrotic area compared with EAT treatment (fixed at 100%). Data are representative of three separate experiments. ^*P<0.01 .

Figure 3. EAT infiltration in human myocardium is associated with myocardial fibrosis.

Red picrosirius staining of left atrial appendage (A) and myocardial (B) sections. High magnification images showing that adipocytes inclusions are associated with myocardial fibrosis (right panels).

Detailed description of the invention:

Mechanisms underlying the effect of EAT on myocardium has been poorly studied in general and even more scarcely in humans. The inventors made the following hypothesis: Because EAT is contiguous with the heart without fascia boundaries, a paracrine process is possible with the release of adipo-fibrokines i.e. adipose produced factors with profibrotic properties. They tested this hypothesis using an original organo-culture model of rat atria in which the histological and structural integrity of the atrial myocardium is maintained during culture. The inventors observed that the secretome from human EAT, but not subcutaneous adipose tissue (SAT), has a marked fibrotic effect on the rat atrial myocardium which appears to be mediated in part by activin A. In situ the inventors confirmed that adipose tissue is in close proximity with the myocardium and is associated with an important fibrosis of human myocardium. The inventors have also found out that inhibiting activin, e.g. by using an anti-activin A antibody, neutralized the pro-fibrotic effects induced by the EAT secretome.

Definitions

"Activin" as used herein, refers to any member of the activin family, regardless of the species of animal with which that member is associated. Activins are part of the TGF-beta family. They exhibit a wide range of biological activities including regulation of cellular proliferation and differentiation. Humans, mice, and rats, as well as many other species are known to produce activins, including activin-A which is a homodimer of beta A monomers; however, other forms of activin, including but not limited to activin-B, C, and E are included herein.. Human Activin- A is a 26 kDa disulfide-linked homodimer of two beta A chains, each containing 1 16 amino acid residues.

In the present invention, by the term "activin inhibitor" is meant a substance that decreases or destroys the physiological function that activin has. It may be the one that directly couples with activin to inhibit the physiological function of activin or may be the one that inhibits the production of activin itself. Alternatively, it may be the one that inhibits the signal transduction generated by the coupling of activin with an activin receptor. The term "substance" refers to any chemically synthesized or naturally derived material or compound.

The "patient" is any mammal, preferably a human subject, including males or females, regardless of his/her body mass index, i.e. regardless of his/her weight. Non-human mammals, e.g. horses, dogs, cats, etc, may also be treated according to the invention. In a particular embodiment the patient shows epicardial fat. In a particular embodiment, patients who show an excess of epicardial adipose tissue (EAT) may particularly benefit from the invention. Epicardial adipose tissue is localized inside the pericardial sac and interacts with myocardium (absence of facia). An "excess" of EAT means a thickness of more than about 5mm, generally between 5 to 10 mm, or between 6 to 10 mm. As used in this specification, the term "about" refers to a range of values ± 10% of the specified value. Preferably, the term "about" refers to a range of values ± 5 % of the specified value.

In a preferred embodiment, the patient shows no atrial fibrillation (AF), in particular no chronic AF, but is diagnosed as being at risk of AF, especially by detecting presence of EAT.

In another embodiment, the patient already shows atrial fibrillation (AF).

The patient may show additional risk factors, such as high blood pressure, obesity, diabetes, age about 75, etc.

"Preventing AF" or "prevention of AF" means that the activin inhibitor reduces the risk of AF or protects the patient against AF. More particularly, the activin inhibitor inhibits the fibrosis of the atrial myocardium. Fibrosis of the atrial myocardium is an interstitial fibrosis. In the context of the invention, prevention of AF includes a prophylactic treatment, including any of: halting the onset, reducing the risk of development, reducing the incidence, delaying the onset, reducing the development, as well as increasing the time to onset of symptoms of AF.

"Treating AF" or "treatment of AF" includes curative treatment. More particularly, curative treatment refers to any of the alleviation, amelioration and/or elimination, reduction and/or stabilization (e.g., failure to progress to more advanced stages) of a symptom, as well as delay in progression of a symptom of AF.

"Atrial fibrillation" includes paroxysmal AF and persistent AF.

Risk of Atrial fibrillation

The invention is most advantageous in patients who are at risk of atrial fibrillation. Such patients more particularly encompass patients who show epicardial fat, preferably patients who show an excess of EAT.

Epicardial fat volume can be measured by using IRM or computed tomography, as described in Obadah Al Chekakie, et al, J Am Coll Cardiol. 2010;56(10):784-788

Echocardiographic measurements and scanner may also be useful to determine thickness of epicardial adipose tissue.

Administration of activin inhibitors

According to the invention, patients at risk of AF are administered with an activin inhibitor. Specific examples of activin inhibitor include follistatin, an anti-activin antibody, an inhibitor to activin receptor or an anti-activin receptor antibody, an inhibitor to signal transduction relating to activin receptor, an activin production or expression inhibitor.

Activins signal through a heteromeric complex of receptor serine kinases which include at least two type I (I and IB) and two type 11 (11 and MB) receptors. An inhibitor of activin receptor includes an antagonist of ACVR1 (type I activin receptor) or ACVR2 (type II activin receptor). The inhibitor of activin receptors preferably bind the activin receptor (i.e. ACVR1 and/or ACVR2) with a high affinity, e.g. with a Kd of less than about 10^~9.

The inhibitor to activin receptor includes proteins or compounds having structures similar to that of follistatin and blocks the activin receptor to inhibit the coupling of activin with follistatin. The activin production or expression inhibitor includes antisense nucleic acid, preferably antisense DNA, to the activin gene, an interferent RNA (iRNA) or a ribozyme. In a particular embodiment, the activin production or expression inhibitor is selected from the group consisting of antisense DNA to the activin gene and an interferent RNA (iRNA).

The term "iRNA" means any RNA which is capable of down-regulating the expression of the targeted gene. It encompasses small interfering RNA (siRNA), double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules. RNAi can comprise naturally occurring RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end of the molecule or to one or more internal nucleotides of the RNAi, including modifications that make the RNAi resistant to nuclease digestion. RNAi may be administered in free (naked) form or by the use of delivery systems that enhance stability and/or targeting, e.g., liposomes, or incorporated into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors (WO 00/53722), or in combination with a cationic peptide (US 2007275923). They may also be administered in the form of their precursors or encoding DNAs. Preferably, the RNAi molecule is a siRNA of at least about 15-50 nucleotides in length, more preferably about 20-25 nucleotides in length. siRNA are usually designed against a region 50-100 nucleotides downstream the translation initiator codon, whereas 5'UTR (untranslated region) and 3'UTR are usually avoided. The chosen siRNA target sequence should be subjected to a BLAST search against EST database to ensure that the only desired gene is targeted. Various products are commercially available to aid in the preparation and use of siRNA. Examples of siRNA inhibiting activin A expression include, but are not limited to, anti-activin A siRNA disclosed in Hoda et al., Br J Cancer. 2012 Dec 4;107(12):1978-86.

Antisense nucleic acid can be complementary to all or part of a sense nucleic acid encoding the target gene product e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence, and it thought to interfere with the translation of the target mRNA. An antisense nucleic acid can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. Particularly, antisense RNA molecules are usually 18-50 nucleotides in length. An antisense nucleic acid for use in the method of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. Antisense nucleic acid may be modified to have enhanced stability, nuclease resistance, target specificity and improved pharmacological properties. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. Ribozyme molecules specific for a gene product can be designed, produced, and administered by methods commonly known to the art (see e.g., Fanning and Symonds, 2006, RNA Towards Medicine (Handbook of Experimental Pharmacology), ed. Springer p. 289-303, reviewing therapeutic use of hammerhead ribozymes and small hairpin RNA).

Other inhibitors can also be used, such as activin neutralizing antibodies, in particular activin neutralizing antibodies which are commercially available, as well as substances, such as "SB- 431542" (4-[4-(1 ,3-benzodioxol-5-yl)-5-(2-pyridyl)-1 H-imidazol-2-yl]benzamide), which is commercially available (Sigma-Aldrich) and interacts with activin receptors.

In an embodiment, the activin inhibitor is an activin neutralizing antibody. Preferably, the activin neutralizing antibody is an antibody directed against activin A, in particular an antibody which is able to bind to activin A and to block or reduce its activity, i.e. to inhibit activin A. More preferably the antibody is able to specifically bind and inhibit activin A.

Examples of activin neutralizing antibodies include, but are not limited to, activin A βΑ subunit antibody (R&D systems, Wiesbaden-Nordenstadt, Germany, Blumensatt et al. Cardiovasc Res. 2013 Nov 1 ;100(2):201 -10) or ACE-01 1 antibody (also named Sotatercept), a chimeric protein containing the extracellular domain of the activin receptor 2A (ACVR2A) fuse to the Fc domain of human igG1 (Raje and Vallet, Curr. Opin. Mol. Ther. 2010 Oct; 12(5):586-97). The term "antibody", as used herein, is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE, and human, humanized or chimeric antibody. In certain embodiments, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and they are most easily manufactured. The term "antibody" is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab') 2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art. A "humanized" antibody is an antibody in which the constant and variable framework region of one or more human immunoglobulins is fused with the binding region, e.g. the CDR, of an animal immunoglobulin. "Humanized" antibodies contemplated in the present invention are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof. Such humanized antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding regions are derived, but to avoid an immune reaction against the non-human antibody. A "chimeric" antibody is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

The action of activin inhibitor can be examined by measuring the activity of activin in the presence or absence of an activin inhibitor. The activity of activin can be measured by in vitro tests using activin A properties to promote fibroblast differentiation into myofibroblast or its capacity to promote atrial fibrosis, as illustrated in the experimental section.

In the present invention, the follistatin is not particularly limited and any follistatin may be used regardless of the type or origin of it as far as it has an activin inhibiting action. For example, as far as it has an activin inhibiting action, it may be the one originated from an animal such as a pig as well as human follistatin, or natural type follistatin or recombinant type follistatin.

It is known that follistatins are glycoproteins whose peptide moiety has a molecular weight of from 30,000 to 40,000 differing in amino acid residue number, e.g., 315, 303, or 288 and in the site and number of attachment of sugar chains. If they have other structural changes than described above, the effect of the present invention can be obtained as far as they have the ability of coupling with activin and retain an equivalent activin inhibitory activity as the activin coupling protein.

Any member of the follistatin family, including but not limited to "FS-300", "FS-288", and other forms as well. See Hoshimoto, et al., J. Biol. Chem., 272(21 ): 13835-43 (1997) incorporated by reference. There is very high conservation of sequences among both the activins and the follistatins, and it is known, that follistatin from one species will inhibit activin from another, different species. Especially preferred are forms of follistatin which bind to cell surface heparin sulphate proteoglycans, such as FS-288.

The natural type follistatin may be prepared by extracting it from an organ of animal, such as an ovary, and subjecting it to a purification step. Recombinant type follistatin can be prepared by introducing human or animal follistatin cDNA into a suitable expression vector, followed by gene transfer into a suitable animal cell with the vector, cultivating the cells and subjecting the culture medium to a purification step.

The recombinant follistatin can be prepared by a method for producing a heterologous protein by an ordinary recombinant technique using a DNA encoding follistatin. The DNA encoding follistatin and the method of producing a recombinant follistatin using the DNA are disclosed in WO89/01945. In the example described hereinbelow, the follistatin used was purified from the culture medium of CHO cells having incorporated therein cDNA corresponding to human follistatin composed of 315 amino acids.

In a preferred embodiment, the activin inhibitor is an anti-activin antibody, especially an activin A neutralizing antibody. Specific antibodies are preferred, i.e. antibodies that show substantially no cross-reaction with any other entity. In another particular embodiment, the antibody may cross-react with other types of activins (e.g. the anti-activin A antibody may show some cross-reactivity with activin B). According to the invention, the activin inhibitor may be administered by any convenient route, especially intravenously. It may also be directly administered or injected into epicardial fat. The pharmaceutical composition comprising the activin inhibitor is formulated in accordance with standard pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known by a person skilled in the art. The pharmaceutical composition may comprise one or several activin inhibitors of the invention. The pharmaceutical composition may also comprise additional active substance(s).

The dosage and route of administration can be determined by any skilled physician, depending on the activin inhibitor and the severity of the AF risk. For example, the activin inhibitor may be administered at a daily dosage of ^g to 100mg.

According to the invention, the activin inhibitor inhibits myocardial remodeling. Myocardial remodeling is manifested clinically as changes in the size, shape, and function of the heart. In a preferred embodiment, the activin inhibitor inhibits myocardial fibrotic remodeling that promotes the occurrence of AF.

The present invention also concerns a method for preventing or treating atrial fibrillation in a patient, which method comprises administering a therapeutically efficient amount of an activin inhibitor in a patient in need thereof, preferably a patient at risk of atrial fibrillation. All the embodiments of the activin inhibitor for use in preventing or treating atrial fibrillation in a patient as disclosed above, are also contemplated in this method. The present invention further concerns the use of an activin inhibitor for the manufacture of a medicament for the prevention or treatment treating atrial fibrillation. All the embodiments of the activin inhibitor for use in preventing or treating atrial fibrillation in a patient as disclosed above, are also contemplated in this aspect. The present invention further concerns an activin inhibitor, for use in preventing or reducing myocardial remodeling, more particularly myocardial fibrotic remodeling, in a patient, especially in a patient at risk of atrial fibrillation. The present invention also concerns a method for reducing myocardial remodeling, in particular myocardial fibrotic remodeling, in a patient, which method comprises administering a therapeutically efficient amount of an activin inhibitor in a patient in need thereof, preferably a patient at risk of atrial fibrillation. Myocardial fibrotic remodeling promotes the occurrence of AF and encompasses abnormal accumulation of collagen fibers secreted by fibroblasts in the extracellular matrix inducing the appearance of fibrous tissue, in particular in the extracellular matrix surrounding myocytes. All the embodiments of the activin inhibitor for use in preventing or treating atrial fibrillation in a patient as disclosed above, are also contemplated in this method.

In another aspect, the present invention further concerns a method for screening or identifying a molecule suitable for treating or preventing atrial fibrillation. The method may be in vivo, ex vivo or in vitro method, preferably in vitro method.

In an embodiment, the method comprises the steps of (i) measuring the activity of activin, preferably activin A, in the presence and absence of candidate molecules and (ii) selecting molecules having the ability to inhibit the activity of activin, preferably activin A. The activity of activin A can be measured by in vitro tests using activin A properties to promote fibroblast differentiation into myofibroblast or its capacity to promote atrial fibrosis.

The below examples illustrate the invention without limiting its scope. Example 1 : Neutralization of Activin A prevents atrial fibrosis and fibrillation MATERIALS AND METHODS

Human Adipose and cardiac Tissue

The inventors obtained paired samples of EAT and SAT from 39 patients undergoing routine cardiac bypass surgery (Table 1 ). 12 paired samples were dedicated to transcriptomic study (Group 1 ) and 27 were dedicated to the study of conditioned media (Group 2). SAT biopsies were taken from the parasternal region, while EAT samples from the left interventricular groove before the cardiopulmonary bypass. In 18 patients, a sample of the right (n=15) or left (n=3) atrial appendage was obtained for histological study. Ventricle human myocardium samples (n=5) were obtained from autopsy.

The ethics committee of Pitie-Salpetriere Hospital approved the clinical investigations. All patients subjected to bypass surgery gave a written informed consent after individual explanation. TABLE 1. Patients characteristics

GROUP 1 GROUP 2

N 12 27

Age 60.1±6.7 65.66±8.6

Sex (M/F) 10/2 18/9

Surgery CABG* CABG*

CAD† (n) 12 27

Diabetes (n) 5 9

Dyslipidemia (n) 10 17

Hypertension (n) 8 12

*CABG, Coronary artery bypass graft

†CAD, Coronary artery disease

Oraanoculture of Adult Rat Atria

In accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health, adult male Wistar rats were anesthetized using sodium pentobarbital, hearts were rapidly excised and left and right atria were dissected and transferred into cold C02-independent medium (Gibco, Invitrogen). The trabeculated myocardium walls were then placed on the porous membranes of inserts filled with M199 culture medium (Gibco, Invitrogen). Atria were cultured during 7 days in standard incubation conditions (37°C, 5% C02).

Adipose tissue-conditioned media was mixed in a proportion of 1 :4 with culture medium. In some experiments, recombinant human/rat Activin A (5 ng/mL, R&D Systems), mouse monoclonal Inhibin βΑ-neutralizing antibody (1 μg mL; Abeam) were added in culture medium Fixed atria were embedded in cryo-conservation medium (Shandon Cryomatrix, Thermo Scientific), frozen with liquid nitrogen, and stored until use at D80°C.

Rat Cardiac Fibroblasts Culture

Atria from three-month-old rats were used to isolate cardiac fibroblast as previously described (Balse et al, Proc Natl Acad Sci U S A 2009;106(34):14681 -6). Next, the cells were cultured in DMEM 4.5 g/L glucose (Gibco) supplemented with 10% SVF, 100 U/mL penicillin, and 100 mg/mL streptomycin, in standard conditions (37°C, 5% C02). Immunohistoloaical Analysis

Frozen sections of rat atria (10 μηι) were stained with Masson's trichrome and picrosirius red for the study of fibrosis which was quantified by histomorphometry using an Alphelys platform (Histolab Software, Plaisir, France) and expressed as the ratio of fibrous tissue area to total tissue surface area (Divoux et al, Diabetes 2010;59(1 1 ):2817-25). Procedure used for the immunofluorescence has been already described (Balse et al, Proc Natl Acad Sci U S A 2009;106(34):14681 -6); the following primary antibodies were used: anti-collagen I (1/200, rabbit polyclonal, Novus Biological), anti-collagen III (1/100, rabbit polyclonal, Abeam), anti- collagen VI (1/100, mouse monoclonal, Abeam) and anti-sarcomeric a-actinin (1/400, mouse monoclonal, Sigma); the following secondary antibodies Alexa Fluor® 488 goat anti-rabbit IgG, Alexa Fluor® 546 goat anti-mouse IgG (1 /400, Invitrogen), and Alexa Fluor® 594 goat anti-mouse IgG (1/400, Molecular Probes, Invitrogen). Nuclei were stained with DAPI (Invitrogen). Multiplexed proteomics and ELISA

Cytometric bead arrays were used to analyze protein composition of EAT and SAT conditioned-media. Multiplex assays were performed according to the manufacturer instructions (R&D Systems, France). Multianalyte profiling was performed on the Luminex-200 system and the Xmap Platform (Luminex Corporation, Austin, TX). Fluorescence data were analyzed with Xponent software by using standard curves obtained from serial dilutions of standard cytokines mixtures. Concentration of Activin A and Follistatin was measured by ELISA assays according to the manufacturer instructions (R&D System, France).

Second Harmonic Generation Imaging of Rat Auricular Fibrosis

Second-harmonic generation (SHG) is an optically nonlinear coherent process used to image the collagen network, based on 2-photon excitation. Multiphoton imaging was performed using 10-μηι thick slices covered with coverslips without any staining.

Statistical Analysis

Values are expressed as mean ± SEM. The Gaussian distribution of all parameters was tested. Differences between variables were determined by the nonparametric Wilcoxon and Mann- Whitney tests for paired and unpaired data, respectively. RESULTS

Epicardial, but Not Subcutaneous, Adipose Tissue Secretome Induces Atrial Fibrosis

EAT was collected in patients with coronary insufficiency, a condition that is associated with biologically active EAT (Mazurek et al, Circulation 2003;108(20):2460-6; Dutour et al, J Clin Endocrinol Metab 2010;95(2):963-7) (Table 1 ). Secretome was obtained by harvesting the conditioned media of adipose tissue samples maintained in culture conditions as previously described 20. To obtain sufficient volume of conditioned culture media, EAT secretomes from two patients were pooled. Incubation of rat atria in organo-culture conditions with EAT but not SAT for one week was associated with the development of epicardial and interstitial fibrosis as evidenced by picrosirius red and Masson's trichrome staining. Picrosirius red staining indicated a 2-fold increase in fibrosis in atria treated with EAT-conditioned (33.78%±1 .33), compared with those incubated with SAT-conditioned (15.33%±1 .60; both P=0.001 ) or control media (15.67%±0.78; both P=0.001 ). Fibrosis was composed of collagens and specifically by collagen types I, III, and VI. We checked that during culture, atrial myocardium preserved its sarcomeric organization and architecture.

Fibroblasts are the primary source of extracellular matrix proteins. Collagen I and a-Smooth Muscle Actin gene expression were up-regulated in atrial fibroblasts treated with EAT- conditioned media during 4 days in culture suggesting their differentiation into myofibroblasts. Collectively, these results indicated that the secretome from EAT induces marked fibrosis of the atrial myocardium ex vivo.

Human Epicardial Adipose Tissue Abundantly Expresses Activin-A

The adipo-cytokines secreted by human EAT were identified using cytometric beads arrays and enzyme-linked immunosorbent assays. EAT secreted angiogenetic factors (VEGF and Thrombospondin-2) and matrix metalloproteinases (MMPs) such as MMP1 , MMP8 and MMP9, more abundantly than SAT.

EAT expressed abundantly Activin A, a member of the TGF-β superfamily. In contrast, TGF- β1 level was not different between EAT and SAT whereas other TGF-β members were even not detectable. There was a 3-fold increase in Activin A concentration in EAT-conditioned media (3560±858.5 pg/mL) compared with SAT-conditioned media (1287±288.6 pg/mL) (p=0.0002). This increase was also confirmed at mRNA and tissue level (Figure 1 ,A through F). Indeed, a trend toward an increase in Activin A mRNA level between EAT and SAT was observed (relative expression, 2.58±0.65 vs. 1 .15±0.28; p=0.07) (Figure 1 A) and a significant greater immunostaining was observed in EAT depot (Figure 1 E and F). In addition, Follistatin, the natural inhibitor of Activin A, was not enhanced in EAT, resulting in an increase in the Activin A/Follistatin ratio at both transcriptional and protein level (Figure 1 , B through D). Given that a pro-fibrotic effect has been ascribed to Activin A in several tissues 21 , we tested the hypothesis that Activin A is involved in EAT-induced atrial fibrosis in subsequent experiments. Activin A Promotes Myocardial Fibrosis

First, after one week of culture of atria with a control medium supplemented with Activin A (5 ng/mL), a marked fibrosis accumulated as observed with EAT secretome (36.36%±2.04 vs. 15.67%±0.78 in controls; P<0.001 ). In addition, Activin A supplementation induced the de novo synthesis of collagen types I, III, and VI (Figure 5, D). Second, we observed that EAT- conditioned media pre-incubated with neutralizing antibodies raised against Activin A failed to induce myocardial fibrosis (EAT+Activin A-neutralizing Ab: 1 1 .83%±0.4; EAT+lgG: 24%±0.93; P=0.002) (Figure 2, A and B). Third, EAT secretome and Activin A induced similar changes in atrial gene expression profile. EAT-conditioned media did not induce additional Activin A expression in atrial organo-culture, suggesting that EAT was the primary source of Activin A. Both EAT and Activin A increased the expression of TGF-βΙ and 2. These results indicated that atrial fibrosis induced by EAT is mainly mediated by Activin A.

EAT is associated with fibrosis of human myocardium

We searched for evidence that in situ the presence of fat depot could be associated with fibrosis of the myocardium. Histological study was performed in left (n=3) and right atrial (n=15) and ventricle (n=5) human myocardium. Figure 3 shows typical example, of dense fat tissue infiltrating the atrial and ventricle myocardium (Figure 3 A and B). This tissue was composed of well organized adipocytes with pericellular fibrosis as described elsewhere 8. At higher magnification, fibrosis could be seen at the interface between adipose and myocardial tissue (Figure 3 A and B). There was also a marked interstitial fibrosis of the neighbouring myocardium (Figure 3 A and B). These results indicate that myocardial fibrosis and EAT can be associated in situ and that there is a close proximity between the two tissue types.

Claims

1 . An activin inhibitor substance, for use in preventing or treating atrial fibrillation in a patient.

2. The activin inhibitor substance for use in preventing atrial fibrillation in a patient according to claim 1 , wherein the patient is at risk of atrial fibrillation.

3. The activin inhibitor substance for use in preventing or treating atrial fibrillation in a patient according to claim 1 or 2, wherein the patient shows epicardial fat.

4. The activin inhibitor substance for use in preventing or treating atrial fibrillation in a patient according to any of claims 1 to 3, wherein the patient shows an excess of epicardial fat.

5. The activin inhibitor substance for use in preventing or treating atrial fibrillation in a patient according to any of claims 1 to 4, wherein the activin inhibitor substance is selected from the group consisting of an anti-activin antibody, follistatin, an inhibitor to activin receptor, an anti- activin receptor antibody and an anti-activin antisense DNA or iRNA.

6. The activin inhibitor substance for use in preventing or treating atrial fibrillation in a patient according to any of claims 1 to 4, wherein the activin inhibitor substance is selected from the group consisting of an anti-activin antibody, follistatin and an inhibitor to activin receptor.

7. The activin inhibitor substance for use in preventing or treating atrial fibrillation in a patient according to any of claims 1 to 4, wherein the activin inhibitor substance is an activin neutralizing antibody.

8. The activin inhibitor substance for use in preventing or treating atrial fibrillation in a patient according to any of claims 1 to 7, wherein the activin inhibitor substance inhibits activin A.

9. The activin inhibitor substance for use in preventing or treating atrial fibrillation in a patient according to any of claims 1 to 7, wherein the activin inhibitor substance is an activin neutralizing antibody that binds and inhibits activin A, preferably that specifically binds and inhibits activin A.

10. The activin inhibitor substance for use in preventing or treating atrial fibrillation in a patient according to any of claims 1 to 9, wherein the activin inhibitor substance is to be administered intravenously.

1 1 . The activin inhibitor substance for use in preventing or treating atrial fibrillation in a patient according to any of claims 1 to 10, wherein the atrial fibrillation is chronic.

12. The activin inhibitor substance for use in preventing or treating atrial fibrillation in a patient according to any of claims 1 to 1 1 , wherein the activin inhibitor substance inhibits myocardial remodeling.

13. The activin inhibitor substance for use in preventing or treating atrial fibrillation in a patient according to any of claims 1 to 1 1 , wherein the activin inhibitor substance inhibits myocardial fibrotic remodeling.