WO2017214684A1

WO2017214684A1 - Methods and products for identifying conditions associated with cardiac fibrotic remodelling

Info

Publication number: WO2017214684A1
Application number: PCT/AU2017/050608
Authority: WO
Inventors: Doan NGO; Aaron SVERDLOV
Original assignee: Adelaide Research & Innovation Pty Ltd
Priority date: 2016-06-17
Filing date: 2017-06-16
Publication date: 2017-12-21

Abstract

The present disclosure relates to methods and products for identifying conditions associated with cardiac fibrotic remodelling. Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling, the method comprising identifying an increased level of follistatin-like 3 in the subject.

Description

METHODS AND PRODUCTS FOR IDENTIFYING CONDITIONS ASSOCIATED WITH CARDIAC FIBROTIC REMODELLING

PRIORITY CLAIM

[001] This application claims priority to Australian provisional patent application number 2016902369 filed on 17 June 2016, the content of which is hereby incorporated by reference.

FIELD

[002] The present disclosure relates to methods and products for identifying conditions associated with cardiac fibrotic remodelling.

BACKGROUND

[003] Biomarkers are emerging as an important tool to assist a clinician with the diagnosis, prognosis and/or screening for a variety of conditions, and also to assist with the clinical management of a patient.

[004] In the cardiovascular field, the prevention and early identification of disease states leads to early treatment with improved outcomes, patient satisfaction, reduced healthcare costs, morbidity and mortality.

[005] The identification of early changes contributing to disease states, and/or the identification of patients at risk, is typically achieved by a combination of good clinical skills and the availability of appropriate diagnostic modalities. The use of biomarkers provides a diagnostic modality that typically is easy to perform and/or inexpensive.

[006] A variety of biomarkers are currently utilised for the assessment of cardiovascular disease risk, such as cholesterol levels, troponin and brain-natriuretic peptides. However, while such biomarkers exist, they are generally only of limited usefulness, because their predictive value is limited and/or their ability to assist with risk stratification is only limited. [007] In addition, some of the markers are not suitable for early detection, as their presence only correlates with latter stages of a condition, and some markers are influenced by other clinical features such as obesity, renal failure and pulmonary disease.

[008] Accordingly, there is a need for new cardiovascular biomarkers. SUMMARY

[009] The present disclosure relates to methods and products for identifying conditions associated with cardiac fibrotic remodelling.

[0010] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling, the method comprising identifying an increased level of folli statin-like 3 in the subject.

[0011] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling, the method comprising:

detecting folli statin-like 3 from the subject; and

identifying the subject as suffering from, or being susceptible to, a cardiac condition associated with cardiac fibrotic on the basis of the level of the folli statin-like 3 detected.

[0012] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling, the method comprising:

detecting folli statin-like 3 from the subject;

using a computer processor means to process data associated with the level of the folli statin-like 3 to generate a likelihood and/or risk of a cardiac condition associated with cardiac fibrotic remodelling occurring in the subject; and identifying the presence or absence of the condition in the subject on the basis of the likelihood and/or risk generated.

[0013] Certain embodiments of the present disclosure provide a method of screening for a condition associated with cardiac fibrotic remodelling occurring in a subject, the method comprising determining the level of follistatin-like 3 in the subject.

[0014] Certain embodiments of the present disclosure provide a method of monitoring progression of a condition associated with cardiac fibrotic remodelling occurring in a subject, the method comprising determining the level of follistatin-like 3 in the subject and determining the extent of disease progression on the basis of the level of follistatin- like 3 so determined.

[0015] Certain embodiments of the present disclosure provide a method of determining the responsiveness of a subject with chronic heart failure to cardiac resynchronisation therapy, the method comprising determining the level of follistatin- like 3 in the subject and determining the responsiveness of the subject to cardiac resynchronisation therapy.

[0016] Certain embodiments of the present disclosure provide a method of determining the likelihood or risk of a condition associated with cardiac fibrotic remodelling occurring in a subject, the method comprising:

determining the level of follistatin-like 3 from the subject; and

determining the likelihood or risk of the subject suffering from, or being susceptible to, a cardiac condition associated with cardiac fibrotic remodelling on the basis of the level of follisttain-3 determined.

[0017] Certain embodiments of the present disclosure provide a kit for performing a method as described herein.

[0018] Certain embodiments of the present disclosure provide a kit when used to identify or screen for a condition associated with cardiac fibrotic remodelling occurring in a subject, the kit comprising one or more reagents for detecting follistatin-like 3. [0019] Certain embodiments of the present disclosure provide a method of preventing and/or treating a condition associated with cardiac fibrotic remodelling in a subject, the method comprising:

determining the level of folli statin-like 3 in the subject; and

treating the subject on the basis of one or more clinical features of the subject and the level of folli statin-like 3 so determined.

[0020] Certain embodiments of the present disclosure provide a method of identifying a subject suitable for treatment for a condition associated with cardiac fibrotic remodelling in a subject, the method comprising:

determining the level of folli statin-like 3 in the subject; and

identifying the subject as a subject suitable for treatment on the basis of one or more clinical features of the subject and the level of folli statin-like 3 so determined.

[0021] Other embodiments are disclosed herein. BRIEF DESCRIPTION OF THE FIGURES

[0022] Certain embodiments are illustrated by the following figures. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the description.

[0023] Figure 1 shows follistatin-like 3 (FSTL3) levels correlate with left ventricular mass as a measure of cardiac hypertrophy. Higher FSTL3 levels predict increased hypertrophy.

[0024] Figure 2 shows in Panel A: FSTL3 levels significantly increase in an incremental fashion in chronic heart failure and acute heart failure patients compared to controls. FSTL3 levels are highest in acute heart failure patients, significantly lower in chronic heart failure patients and lowest in normal controls. Panel B: FSTL3 levels are significantly higher in acute versus chronic heart failure patients; the current gold- standard biomarker NT-proBNP levels are not significantly different between these 2 groups and thus cannot be used to discriminate between them.

[0025] Figure 3 shows in Panel A: FSTL3 levels decrease rapidly in acute heart failure patients in response to 5 weeks of anti -failure treatment. Panel B: NT-proBNP levels (current gold standard for monitoring treatment response) are still unchanged at 5 weeks of anti-failure treatment in the same cohort.

[0026] Figure 4 shows that in patients with bicuspid aortic valve FSTL3 levels correlate with A) ascending aortic diameter; B) Aortic sinus diameter; and C) Sino- tubular diameter. Higher FSTL3 levels predict higher diameter at all 3 levels of measurement.

[0027] Figure 5 shows in Panel A: Baseline FSTL3 levels inversely correlate with improvements in 6 minute walk test (6MWT) after implantation of cardiac resynchronisation therapy. Panel B: Baseline FSTL3 levels inversely correlate with improvements in maximal oxygen consumption (V02 max) after implantation of cardiac resynchronisation therapy. Higher FSTL3 levels prior to implantation of cardiac resynchronisation therapy predict poorer physical performance as assessed by both 6MWT and V02 max at 6 months post-implant.

[0028] Figure 6 shows that FSTL3 levels are higher in patients with new onset AF versus those with chronic AF.

[0029] Figure 7 shows in Panel A: Galectin-3 levels are higher in patients with heart failure compared to healthy older individuals. Panel B: Baseline Galectin-3 levels inversely correlate with improvements in left ventricular end-systolic and end-diastolic volumes after implantation of cardiac resynchronisation therapy. Higher baseline Galectin-3 levels predict ongoing increase/lack of reduction in these volumes and thus worse response to cardiac resynchronisation therapy as assessed echocardiographically.

[0030] Figure 8 shows in Panel A: Galectin-3 levels are higher in patients with new onset AF versus those with chronic AF. In patients with AF, Galectin-3 levels correlate with B) C-reactive protein (a marker of inflammation); C) NT-proBNP (a marker of atrial stretch); and D) CHADS2VASC scores (a clinical stroke risk score) [0031] Figure 9 shows that Galectin-3 levels significantly increase in an incremental fashion in chronic heart failure and acute heart failure patients compared to controls. Galectin-3 levels are highest in acute heart failure patients, significantly lower in chronic heart failure patients and lowest in normal controls.

[0032] Figure 10 shows patients with FSTL3 and galectin-3 levels in the lowest quartiles had better response to cardiac resynchronisation therapy, as assessed by both 6MWT and V0₂max, compared to those in the highest quartiles.

[0033] Figure 11 shows patients with FSTL3 and Galectin-3 levels in the lowest quartiles had better response to cardiac resynchronisation therapy, as assessed by both change in left ventricular end-systolic and end-diastolic volumes, compared to those in the highest quartiles.

[DETAILED DESCRIPTION

[0034] The present disclosure relates to methods and products for identifying conditions associated with cardiac fibrotic remodelling.

[0035] The present disclosure is based, at least in part, on the identification of two highly sensitive biomarkers of early sub-clinical cardiac dysfunction, namely follistatin- like 3 (FSTL-3) and galectin-3. It has been found that both of the polypeptides are elevated in plasma of human subjects with echocardiographically detectable but asymptomatic left ventricular hypertrophy. Both markers are elevated prior to any elevation of troponin or BNP, which are the more conventional markers used for detection of cardiac dysfunction.

[0036] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling.

[0037] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling, the method comprising identifying an increased level of folli statin-like 3 in the subject. [0038] In certain embodiments, the subject is a human subject.

[0039] In certain embodiments, the subject is a mammalian subject. In certain embodiments, the subject is an animal. In certain embodiments, the subject is a livestock animal (such as a horse, a cow, a sheep, a goat, a pig), a domestic animal (such as a dog or a cat) or other type of animal such as a primate, a rabbit, a rat, a mouse, a bird and a laboratory animal. Applications of the present disclosure in animals are contemplated.

[0040] In certain embodiments, the subject has an increased risk or likelihood of suffering from, or being susceptible to, a condition associated with cardiac fibrotic remodelling.

[0041] In certain embodiments, the subject does not show any increase in the level of troponin and/or BNP at the time of determining the level of follistatin-like 3. Methods for determining the level of troponin and/or BNP are known in the art.

[0042] Methods for detecting FSTL-3 and determining the level of FSTL-3 are known in the art.

[0043] In this regard, the determining of the level of a marker as described herein may, for example, refer to determining the level of the protein form of a marker and/or a RNA form of the markers, such as a mRNA.

[0044] In certain embodiments, the level of FSTL-3 is compared to a level of the marker that is present in a subject not suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling. In certain embodiments, the level of FSTL- 3 is compared to a level of the marker that is a level of the marker in the general population. In certain embodiments, the level of FSTL-3 is compared to a level of the marker that is indicative of a subject not suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling. In certain embodiments, the level of FSTL- 3 is compared to a level of the marker that is indicative of a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling.

[0045] In certain embodiments, the level of FSTL-3 indicative of a condition associated with cardiac fibrotic remodelling is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 800%, at least 850%, at least 900%, at least 950%, or at least 1000%). Other levels are contemplated.

[0046] In certain embodiments, the level of FSTL-3 indicative of a condition associated with cardiac fibrotic remodelling is increased by at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.5 fold, at least 3.0 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold, at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0 fold, or at least 10 fold. Other levels are contemplated.

[0047] For example, the median value for FSTL-3 measured in plasma in healthy ageing patients is 5600 + 956 pg/ml, with a typical range from about 3840 to 8160 pg/ml.

[0048] In this regard, wherever a value for the level for a plasma marker is provided herein, it is to be understood that a similar level is anticipate for the level of the maker in blood or serum.

[0049] In certain embodiments, the level of FSTL-3 indicative of a condition associated with cardiac fibrotic remodelling comprises a level of greater than 5500 pg/ml, greater than 6000 pg/ml, greater than 6500 pg/ml, greater than 6600 pg/ml, greater than 7000 pg/m, greater than 7500 pg/ml, greater than 8000 pg/ml, greater than 8500 pg/ml, greater than 8600 pg/ml, greater than 9000 pg/ml, greater than 9500 pg/ml, greater than 9600 pg/ml, greater than 10,000 pg/ml, greater than 10500 pg/ml, greater than 11000 pg/ml, greater than 11500 pg/ml, greater than 12000 pg/ml. greater than 12500 pg/ml, greater than 13000 pg/m, greater than 13500 pg/ml, greater than 14000 pg/ml, greater than 14500 pg/ml, or greater than 15000 pg/ml/. Other levels are contemplated. [0050] In certain embodiments, the method comprises determining the level of FSTL- 3 protein. In certain embodiments, the method comprises determining the level of a FSTL-3 RNA, such as a mRNA. Methods for detecting proteins and RNAs are known in the art.

[0051] In certain embodiments, the method comprises determining the level of FSTL- 3 by an immunological method, such as ELISA or Western analysis. In certain embodiments, the method comprises determining the level of FSTL-3 by an immunosorbent assay. Other methods are contemplated. For example, methods that measure the level of transcription of a gene for FSTL-3 (FSTL3; eg Genbank Accession No. NM_005860 in the human) may be utilised. Methods for determining the level of expression (protein and transcription are known in the art).

[0052] In certain embodiments, the determination of the level of FSTL-3 comprises use of an agent that detects FSTL-3. In certain embodiments, the determination of the level of FSTL-3 comprises the use of an agent that binds to FSTL-3. In certain embodiments, the agent is an anti-FSTL3 antibody, and/or an antigen binding part thereof. Use of other types of agents is contemplated.

[0053] In certain embodiments, the method comprises detecting FSTL-3 by an immunological method. Products for detection and determination of the concentration of FSTL-3 are commercially available. In certain embodiments, the method comprises determining the level of FSTL-3 by ELISA.

[0054] In certain embodiments, the method comprises detecting FSTL-3 and one or more other markers. Suitable methods for detecting other marker(s) may be selected.

[0055] In certain embodiments, the method comprises detecting FSTL-3 and one or more other markers, such as troponin and/or BNP. In certain embodiments, the method comprises determining the level of FSTL-3 and one or more other markers, such as troponin and/or BNP. Methods for determining the level of troponin and BNP are known in the art.

[0056] In certain embodiments, the method comprises determining the level of galectin-3. In certain embodiments, the method comprises determining the level of FSTL-3 and galectin-3.

[0057] In certain embodiments, the method comprises identifying an increased level of galectin-3 in the subject.

[0058] Methods for detecting galectin-3 and determining the level of galectin-3 are known in the art.

[0059] In certain embodiments, the method comprises determining the level of galactin-3 protein. In certain embodiments, the method comprises determining the level of a galactin-3 RNA, such as a mRNA.

[0060] In certain embodiments, the method comprises determining the level of galectin-3 by an immunological method, such as ELISA or Western analysis. In certain embodiments, the method comprises determining the level of galectin-3 by an immunosorbent assay. Other methods are contemplated. For example, methods that measure the level of transcription of a gene for galectin-3 (LGALS3; eg Genbank Accession No. NM_001177388.1, NM_002306.3 in the human) may be utilised.

[0061] In certain embodiments, the determination of the level of galectin-3 comprises use of an agent that detects galectin-3. In certain embodiments, the determination of the level of galectin-3 comprises the use of an agent that binds to galectin-3. In certain embodiments, the agent is an anti-galectin-3 antibody, and/or an antigen binding part thereof. Other types of agents are contemplated.

[0062] In certain embodiments, the method comprises detecting galectin-3 by an immunological method. In certain embodiments, the method comprises determining the level of galectin-3 by an immunological method. Products for detection and determination of the concentration of galectin-3 are commercially available. In certain embodiments, the method comprises determining the level of galectin-3 by ELISA.

[0063] In certain embodiments, the level of galectin-3 is compared to a level of the marker that is present in a subject not suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling. In certain embodiments, the level of galectin-3 is compared to a level of the marker that is a level of the marker in the general population. In certain embodiments, the level of galectin-3 is compared to a level of the marker that is indicative of a subject not suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling. In certain embodiments, the level of galectin-3 is compared to a level of the marker that is indicative of a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling.

[0064] In certain embodiments, the method comprises determining the level of FSTL- 3 directly in vivo.

[0065] In certain embodiments, the method comprises determining the level of FSTL- 3 in a biological sample.

[0066] In certain embodiments, the method comprises obtaining a biological sample from the subject and determining the level of FSTL-3 and/or one or more other markers. The term "sample" refers to a sample obtained from a subject, or any derivative, extract, concentrate, mixture, or otherwise processed form thereof.

[0067] Methods for obtaining biological samples are known in the art. Examples of biological samples include biological fluids, blood samples, plasma samples, serum samples, urine samples, tear samples, saliva samples, swab samples, hair samples, skin samples, dried blood samples, dried samples on a matrix, a biopsy, and fecal samples. Methods for collecting biological samples are known in the art. For example, blood samples may be collected using standard techniques such as drawing blood into a collection tube, pin-prick followed by absorption onto filter paper (eg Guthrie card), and devices that withdraw blood using a vacuum (eg Hemolink, Tasso Inc.).

[0068] In certain embodiments, the biological sample comprises one or more of blood, plasma and serum.

[0069] In certain embodiments, the FSTL-3 is present in a biological fluid. In certain embodiments, the FSTL-3 is a blood marker, a plasma marker and/or a serum marker.

[0070] In certain embodiments, the method comprises processing the biological sample to allow detection of a marker(s). In certain embodiments, the method comprises processing a biological sample obtained from the subject and detecting the marker(s). In certain embodiments, the method comprises processing the biological sample to allow detection marker(s) in the biological sample.

[0071] In certain embodiments, the method comprises obtaining a biological sample from the subject and processing the sample to allow detection of folli statin-like 3 and/or one or more other markers.

[0072] In certain embodiments, the method comprises contacting FSLT-3 with an anti- FSTL-3 antibody.

[0073] In certain embodiments, the method comprises obtaining a biological sample from the subject, optionally processing the sample to allow detection of FSTL-3 if required, contacting the biological sample with an anti-FSTL-3 antibody and detecting binding between the FSTL-3 and the antibody.

[0074] In certain embodiments, the method comprises obtaining a plasma, serum or blood sample from the subject, optionally processing the sample to allow detection of FSTL-3 if required, contacting the sample with an anti-FSTL-3 antibody and detecting binding between the FSTL-3 and the antibody.

[0075] In certain embodiments, the method comprises assessing one or more clinical features of the subject. Examples of clinical features are described herein.

[0076] In certain embodiments, the method comprises identifying an increased level of FSLT-3 and assessing one or more clinical features.

[0077] In certain embodiments, the condition associated with cardiac fibrotic remodelling is selected from one of cardiac hypertrophy, heart failure, response to cardiac resynchronisation therapy, paroxysmal atrial fibrillation, aortic root dilatation with aortic valve disease, chemotherapy-induced cardiovascular toxicity and stroke arising from atrial fibrillation. Other conditions are contemplated.

[0078] In certain embodiments, the condition is selected from one or more of coronary heart disease, myocardial infarction and cardiovascular disease. [0079] In certain embodiments, the method comprises identifying an increased level of FSLT-3 and the presence of one or more clinical features associated with the condition.

[0080] In certain embodiments, the method comprises detecting FSTL-3 and one or more other markers, such as troponin and/or BNP. Methods for determining the level of troponin and BNP are known in the art.

[0081] In certain embodiments, the method comprises using a computer processor means to process data associated with the level of the follistatin-like 3. Use of a computer processor means to process data are known in the art, utilising suitable algorithms.

[0082] In certain embodiments, the method comprises using a computer processor means to process data associated with the level of the follistatin-like 3 to generate a likelihood and/or risk of a cardiac condition associated with cardiac fibrotic remodelling occurring in the subject.

[0083] In certain embodiments, the condition associated with cardiac fibrotic remodelling is cardiac hypertrophy.

[0084] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to, cardiac hypertrophy, the method comprising identifying an increased level of follistatin-like 3 in the subject.

[0085] In this regard, it has been recognised that that circulating FSTL3 levels are predictive of human myocardial hypertrophy, and in particular that the levels of FSTL3 are predictive of cardiac hypertrophy prior to development of clinical symptoms.

[0086] Cardiac hypertrophy is the precursor to a multitude of disease states and in itself is independently associated with increased cardiovascular morbidity and mortality. Cardiac hypertrophy develops in response to many factors or stimuli including age, oxidative stress, diabetes, smoking, hypertension, renal disease, and inflammation to name a few. Cardiac hypertrophy leads to diastolic and then systolic heart failure, atrial fibrillation, renal impairment, increased risk of heart attacks and strokes, and worse outcomes in all these conditions. [0087] Currently the only definitive way to diagnose cardiac hypertrophy is by echocardiography or cardiac MRI which is both time-consuming and expensive. Furthermore, especially in the case of echocardiography, it can only pick up cardiac hypertrophy once a significant increase in wall thickness has occurred (because of technical limitations), while cardiac MRI is only available in select centres and is not indicated for screening for hypertrophy in the general population.

[0088] Treatment options exist that have been shown to improve outcomes in patients with cardiac hypertrophy and certain comorbidities. The earlier is treatment is initiated, the better the response to cardiac hypertrophy and thus the outcome. It is preferable to identify a subject with hypertrophy before any significant hypertrophy develops. Treatments for cardiac hypertrophy are known in the art.

[0089] For this reason, in certain embodiments FSTL-3 may be used to identify subjects with early changes or at risk of developing cardiac hypertrophy. This allows stratification of patients according to their risk of developing cardiac hypertrophy.

[0090] Patients with appropriate risk factors, further deemed to be high risk on the basis of their biomarker profile can then undergo more extensive/definitive investigations and/or be started on preventative therapy depending on the clinical scenario and thus many complications of cardiac hypertrophy may be potentially be avoided.

[0091] In certain embodiments, the subject has an increased risk or likelihood of suffering from, or being susceptible to, cardiac hypertrophy.

[0092] In certain embodiments, the method comprises identifying an increased level of FSLT-3 and identifying one or more clinical features associated with cardiac hypertrophy.

[0093] In certain embodiments, the clinical features comprise clinical features associated with the presence and/or absence of cardiac hypertrophy, and/or clinical features associated with the progression of cardiac hypertrophy. Clinical features are known in the art, such as clinical features associated with left ventricular hypertrophy of various morphologies, hemodynamic abnormalities, LV outflow obstruction, diastolic dysfunction, myocardial ischemia and mitral regurgitation.

[0094] In certain embodiments, a plasma level of folli statin-like 3 greater than 6500 pg/ml is indicative that the subject is suffering from, or susceptible to, cardiac hypertrophy.

[0095] In certain embodiments, a plasma level of folli statin-like 3 greater than 5500 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, cardiac hypertrophy.

[0096] In certain embodiments, a plasma level of follistatin-3 in the range from 5500 to 38000 pg/ml is indicative that the subject is suffering from, or susceptible to, cardiac hypertrophy. In certain embodiments, a plasma level of follistatin-3 in the range from 8000 to 38000 pg/ml is indicative that the subject is suffering from, or susceptible to, cardiac hypertrophy. In certain embodiments, a plasma level of follistatin-3 in the range from 8200 to 38000 pg/ml is indicative that the subject is suffering from, or susceptible to, cardiac hypertrophy. Other ranges are contemplated.

[0097] In certain embodiments, a plasma level of follistatin-3 in the range from 6500 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, cardiac hypertrophy. In certain embodiments, a plasma level of follistatin-3 in the range from 8000 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, cardiac hypertrophy. In certain embodiments, a plasma level of follistatin-3 in the range from 8200 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, cardiac hypertrophy. Other ranges are contemplated.

[0098] In certain embodiments, the method comprises determining the level of one or more other markers.

[0099] In certain embodiments, the method comprises the level of galectin-3. In certain embodiments, the method comprises determining the level of FSTL-3 and galectin-3.

[00100] In certain embodiments, the method comprises identifying an increased level of galectin-3 in the subject.

[00101] In certain embodiments, the method comprises determining the level of one or more of N-terminal pro-brain natriuretic peptide, mid-regional pro-atrial natriuretic peptide, mid-regional pro-adrenomedullin, C-terminal pro-endothelin, osteoprotegerin and soluble ST-2. Methods for using such markers are known in the art, for example as described in Coutinho et al. (2011) Hypertension 58: 920-925.

[00102] In certain embodiments, the method comprises identifying an increased level of FSLT-3 in combination with one or more other markers and/or clinical features, such as one or more clinical features associated with cardiac hypertrophy.

[00103] In certain embodiments, the condition associated with cardiac fibrotic remodelling is heart failure (HF).

[00104] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to, heart failure, the method comprising identifying an increased level of folli statin-like 3 in the subject.

[00105] In this regard, certain embodiments of the present disclosure are based, at least in part, that plasma levels of FSTL-3 are increased in HF patients, and that acute HF patients appear to have incremental increase in FSTL-3 levels compared to those at chronic state, independent of NT-proBNP differences. These results indicate that FSTL- 3 is a biomarker to detect changes in HF, prior to changes in NT-proBNP. Treatment options for heart failure are known in the art.

[00106] In certain embodiments, the heart failure is acute heart failure. In certain embodiments, the heart failure is chronic heart failure.

[00107] In certain embodiments, the subject has an increased risk or likelihood of suffering from, or being susceptible to, heart failure. [00108] In certain embodiments, the subject has an increased risk or likelihood of suffering from, or being susceptible to, acute heart failure.

[00109] In certain embodiments, the subject has an increased risk or likelihood of suffering from, or being susceptible to, chronic heart failure.

[00110] In certain embodiments, the subject does not show any increase in the level of NT-proBNP at the time of determining the level of folli statin-like 3. Methods for determining the level of NT-proBNP are known in the art.

[00111] In certain embodiments, the method comprises identifying an increased level of FSLT-3 in combination with one or more clinical features.

[00112] In certain embodiments, the clinical features comprise clinical features associated with the presence and/or absence of heart failure, and/or clinical features associated with the progression of heart failure. Clinical features are known in the art, such as worsening exertional breathlessness, paroxysmal nocturnal dyspnoea and orthopnoea.

[00113] In certain embodiments, a plasma level of folli statin-like 3 greater than 7000 pg/ml is indicative that the subject is suffering from, or susceptible to, heart failure.

[00114] In certain embodiments, a plasma level of folli statin-like 3 greater than 6500 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, heart failure.

[00115] In certain embodiments, a plasma level of folli statin-like 3 greater than 10000 pg/ml is indicative that the subject is suffering from, or susceptible to, acute heart failure.

[00116] In certain embodiments, a plasma level of folli statin-like 3 greater than 9000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, acute heart failure.

[00117] In certain embodiments, a plasma level of folli statin-like 3 greater than 7000 pg/ml is indicative that the subject is suffering from, or susceptible to, chronic heart failure.

[00118] In certain embodiments, a plasma level of folli statin-like 3 greater than 6500 pg/ml is indicative that the subject has an increased likelihood or risk of suffering from, or being susceptible to, chronic heart failure.

[00119] In certain embodiments, a plasma level of follistatin-3 in the range from 7000 to 38000 pg/ml is indicative that the subject is suffering from, or susceptible to, heart failure. In certain embodiments, a plasma level of follistatin-3 in the range from 8000 to 38000 pg/ml is indicative that the subject is suffering from, or susceptible to, heart failure. In certain embodiments, a plasma level of follistatin-3 in the range from 8200 to 38000 pg/ml is indicative that the subject is suffering from, or susceptible to, heart failure. Other ranges are contemplated.

[00120] In certain embodiments, a plasma level of follistatin-3 in the range from 6500 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, heart failure. In certain embodiments, a plasma level of follistatin-3 in the range from 8000 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, heart failure. In certain embodiments, a plasma level of follistatin-3 in the range from 8200 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, heart failure. Other ranges are contemplated.

[00121] In certain embodiments, a plasma level of follistatin-3 in the range from 10000 to 38000 pg/ml is indicative that the subject is suffering from, or susceptible to, acute heart failure. Other ranges are contemplated.

[00122] In certain embodiments, a plasma level of follistatin-3 in the range from 9000 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, acute heart failure. Other ranges are contemplated.

[00123] In certain embodiments, a plasma level of follistatin-3 in the range from 7000 to 38000 pg/ml is indicative that the subject is suffering from, or susceptible to, chronic heart failure. In certain embodiments, a plasma level of follistatin-3 in the range from 8000 to 38000 pg/ml is indicative that the subject is suffering from, or susceptible to, chronic heart failure. In certain embodiments, a plasma level of follistatin-3 in the range from 8200 to 38000 pg/ml is indicative that the subject is suffering from, or susceptible to, chronic heart failure. Other ranges are contemplated.

[00124] In certain embodiments, a plasma level of follistatin-3 in the range from 6500 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, chronic heart failure. In certain embodiments, a plasma level of follistatin-3 in the range from 8000 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, chronic heart failure. In certain embodiments, a plasma level of follistatin-3 in the range from 8200 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, chronic heart failure. Other ranges are contemplated.

[00125] In certain embodiments, the method comprises determining the level of one or more other markers.

[00126] In certain embodiments, the method comprises determining the level of galectin-3. In certain embodiments, the method comprises determining the level of FSTL-3 and galectin-3.

[00127] In certain embodiments, the method comprises identifying an increased level of galectin-3 in the subject.

[00128] In certain embodiments, the method comprises determining the level of one or more B P and NT-proB P, MR-proA P, MR-proADM, troponins, sST2, and GDF- 15. Methods for using such markers are known in the art, for example as described in Gaggin et al. (2013) Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1832(12): 2442-2450.

[00129] In certain embodiments, the condition associated with cardiac fibrotic remodelling is response to cardiac resynchronisation therapy.

[00130] Certain embodiments of the present disclosure provide a method of identifying a subject responsive to cardiac resynchronisation therapy, the method comprising identifying an increased level of folli statin-like 3 in the subject.

[00131] Cardiac Resynchronisation Therapy (CRT) is in common clinical use as a component of the management of chronic heart failure. Apart from medications, CRT decreases the combined risk of death from any cause or first hospitalization and, when combined with an implantable defibrillator, significantly reduces mortality. That being said, up to 30-50% of patients with severe systolic heart failure and implanted with CRT device derive no benefits, despite stringent criteria for CRT implantation. To date, the pathophysiology of this phenomenon remains unknown.

[00132] Strategies to reduce healthcare costs and lead to better outcomes in patients with advanced heart failure needing CRT are lacking. While there are some costly imaging modalities (MRI) available to potentially help optimize CRT therapy, even they have not demonstrated consistent effectiveness. There is a lack of tests that can predict success of CRT prior to implantation.

[00133] In this regard, certain embodiments of the present disclosure are based, at least in part, on the finding that FSTL-3 levels can be used to predict improvement in cardiorespiratory function in patients with severe heart failure after cardiac resynchronization therapy (CRT) device insertion. This provides an innovative and cost effective way to risk stratify patients and predict the success of planned CRT implantation.

[00134] In certain embodiments, the method comprises identifying an increased level of FSLT-3 in combination with one or more clinical features, such as one or more clinical features associated with chronic heart failure.

[00135] In certain embodiments, the method comprises determining the level of one or more other markers.

[00136] In certain embodiments, the method comprises determining the level of galectin-3. In certain embodiments, the method comprises determining the level of FSTL-3 and galectin-3.

[00137] In certain embodiments, the method comprises identifying an increased level of galectin-3 in the subject.

[00138] In certain embodiments, a plasma level of folli statin-like 3 of greater than 130 pg/ml is indicative that the subject has a decreased likelihood of being responsive to cardiac resynchronisation therapy.

[00139] In certain embodiments, a plasma level of folli statin-like 3 of less than 10000 pg/ml is indicative that the subject has an increased likelihood of being responsive to cardiac resynchronisation therapy.

[00140] In certain embodiments, a plasma level of galectin-3 of greater than 15 ng/ml is indicative that the subject has a decreased likelihood of being responsive to cardiac resynchronisation therapy, and a plasma level of galectin-3 of less than 12.5 ng/ml is indicative that the subject has an increased likelihood of being responsive to cardiac resynchronisation therapy.

[00141] Methods for assessing the ability of a subject to respond to cardiac resynchronisation therapy are known in the art, such as cardiac function after CRT, performance in a walk test after CRT and maximum oxygen consumption after CRT.

[00142] In certain embodiments, the condition associated with cardiac fibrotic remodelling is paroxysmal atrial fibrillation and/or stroke arising from atrial fibrillation.

[00143] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to paroxysmal atrial fibrillation, the method comprising identifying an increased level of folli statin-like 3 in the subject.

[00144] Atrial fibrillation (AF) is the commonest arrhythmia. The major complication of AF is stroke due to increased thromboembolic risk engendered by AF. As many as 35% of all strokes are caused by AF. [00145] While some patients can be diagnosed with AF rapidly, a substantial proportion (over 50%) present with intermittent symptoms and are in sinus (normal) rhythm at clinical review, thus delaying the diagnosis and critical therapy (sometimes by months or years, with inherent increase in stroke risk in the interim). Most of these patients present with intermittent palpitations or similar symptoms and the aggressiveness of investigations depends purely on clinical judgement of the physician and adequacy of the history from the patient.

[00146] Identification of biomarkers of early/new/recent onset/episodes of AF would greatly streamline this process, by risk stratifying patients into high/increased risk of episode of AF versus low risk. Higher risk patients may undergo expeditious/extensive monitoring and investigations leading to early definitive diagnosis and treatment, while low risk patients would undergo routine/non-urgent investigations resulting in better healthcare cost utilization without compromising patients' outcomes.

[00147] In this regard, certain embodiments of the present disclosure are based, at least in part, on the recognition that recent onset of AF is associated with elevated FSTL-3 levels. This indicates that FSTL-3 may be a new biomarker in AF. The extent of elevation of FSTL-3 levels in new onset AF correlates with parameters of stretch, inflammation and elevation of CHADSs score. Early elevation of FSTL-3 in new onset AF may be the precipitant for the inflammatory activation leading to development of long-term myocardial fibrosis in chronic AF.

[00148] In certain embodiments, the method comprises identifying an increased level of FSLT-3 in combination with one or more clinical features, such as clinical features associated with AF.

[00149] In certain embodiments, the clinical features comprise clinical features associated with the presence and/or absence of AF, and/or clinical features associated with the progression of AF. Clinical features are known in the art.

[00150] In certain embodiments, the method comprises determining the level of galectin-3. In certain embodiments, the method comprises determining the level of FSTL-3 and galectin-3. [00151] In certain embodiments, the method comprises identifying an increased level of galectin-3 in the subject.

[00152] In certain embodiments, the method comprises determining the level of one or other markers.

[00153] In certain embodiments, the method comprises determining the level of one or more of B P, NT-proB P and troponins. Methods for using such markers are known in the art, for example as described in Bugnicourt et al (2010) European Neurology, vol. 63, no. 1, pp. 24-28, 2010 and Wozakowska-Kaplon B. (2004) The American Journal of Cardiology, vol. 93, no. 12, pp. 1555-1558, 2004.

[00154] In certain embodiments, a plasma level of folli statin-like 3 greater than 9000 pg/ml is indicative that the subject is suffering from, or susceptible to, paroxysmal atrial fibrillation.

[00155] In certain embodiments, a plasma level of folli statin-like 3 greater than 6600 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or susceptible to, paroxysmal atrial fibrillation.

[00156] In certain embodiments, a plasma level of follistatin-3 in the range from 9000 to 38000 pg/ml is indicative that the subject is suffering from, or susceptible to, paroxysmal atrial fibrillation. Other ranges are contemplated.

[00157] In certain embodiments, a plasma level of follistatin-3 in the range from 6600 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, paroxysmal atrial fibrillation. In certain embodiments, a plasma level of follistatin-3 in the range from 8000 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, paroxysmal atrial fibrillation. In certain embodiments, a plasma level of follistatin-3 in the range from 8200 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, paroxysmal atrial fibrillation. Other ranges are contemplated.

[00158] Furthermore, certain embodiments of the present disclosure are based on the recognition that FSTL-3 correlates with clinical score (CHADS2-VASC) for predicting stroke risk and thus may be used in certain embodiments as an adjunct to this clinical score to further individualize a person's stoke risk and personalize treatment.

[00159] Certain embodiments of the present disclosure provide a method of identifying a subject susceptible to stroke or stroke arising from atrial fibrillation, the method comprising identifying an increased level of follistatin-like 3 in the subject.

[00160] In certain embodiments, the method comprises identifying an increased level of FSLT-3 and the use of one or more clinical features, such as one or more clinical features associated with atrial fibrillation.

[00161] In certain embodiments, the clinical features comprise clinical features associated with the presence and/or absence of AF and/or clinical features associated with the progression of AF.

[00162] In certain embodiments, a plasma level of follistatin-like 3 greater than 9600 pg/ml is indicative that the subject is susceptible to stroke arising from atrial fibrillation.

[00163] In certain embodiments, a plasma level of follistatin-like 3 greater than 8600 pg/ml is indicative that the subject has an increased risk or likelihood of being susceptible to stroke arising from atrial fibrillation.

[00164] In certain embodiments, a plasma level of follistatin-3 in the range from 9600 to 38000 pg/ml is indicative that the subject is suffering from, or susceptible to, stroke arising from atrial fibrillation. Other ranges are contemplated.

[00165] In certain embodiments, a plasma level of follistatin-3 in the range from 8600 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, stroke arising from atrial fibrillation. Other ranges are contemplated.

[00166] In certain embodiments, the method comprises determining the level of galectin-3. In certain embodiments, the method comprises determining the level of FSTL-3 and galectin-3. [00167] In certain embodiments, the method comprises identifying an increased level of galectin-3 in the subject.

[00168] In certain embodiments, the method comprises determining the level of one or other markers.

[00169] In certain embodiments, the method comprises determining the level of one or more of B P, NT-proB P and troponins. Methods for using such markers are known in the art, for example as described in Bugnicourt et al (2010) European Neurology, vol. 63, no. 1, pp. 24-28, 2010 and Wozakowska-Kaplon B. (2004) The American Journal of Cardiology, vol. 93, no. 12, pp. 1555-1558, 2004.

[00170] In certain embodiments, the condition associated with cardiac fibrotic remodelling is aortic root dilatation.

[00171] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to, aortic root dilatation, the method comprising identifying an increased level of folli statin-like 3 in the subject.

[00172] This embodiment of the present disclosure is based, at least in part, on the finding that patients with bicuspid aortic valve (BAV) have increased FSTL-3 levels. FSTL-3 levels appear to be exclusively associated with aortopathy in BAV rather than a predictor of aortic valve hemodynamic changes. Accordingly, strategies that aim to slow aortopathy in BAV may be able to take into account FSTL-3 levels as part of the management of BAV-associated complications.

[00173] Aortic valve disease encapsulates 2 entities - stenosis (narrowing of the valve) and regurgitation (backward leakage through the valve due to structural deformation/dysfunction).

[00174] Aortic valve stenosis is the commonest form of valvular heart disease in Western world. It can occur in previously structurally normal valves, but occurs (to some degree almost universally) in those with bicuspid aortic valve (BAV). BAV is the commonest congenital form of heart disease and occurs in 0.5 - 2% of population. Aortic valve regurgitation is not as common in previously normal valves with increasing incidence in those with BAV.

[00175] Aortic root dilatation is an uncommon but potentially life threatening complication of aortic valve disease. In patients with structurally normal valves, it is usually associated with increasing severity of aortic valve regurgitation. In patients with BAV, on the other hand, it is much more common and can occur with both valve stenosis and regurgitation and does not necessarily track with severity of valvular lesion and commonly accompanies even mild-to-moderate stenosis and thus requires separate monitoring. The main complications of aortic root dilatation are dissection or rupture that can lead to rapid death or major adverse outcomes. The only management option is surgical.

[00176] Patients with aortic valve disease are monitored with serial echocardiograms at certain time intervals. Whilst this works well for assessments of severity of valve disease, it may not be adequate for assessment of aortic root/ascending aortic diameter. Currently, gold standard modalities to assess these include either CT angiography or MRI angiography (MRA). For both of these modalities there are substantial costs (MRA > CT), radiation exposure (CT), contrast exposure (CT & MRA) and risk of renal dysfunction (CT) or nephrogenic systemic fibrosis (MRA). Thus, these are not used for routine screening, but only for definitive assessment if clinical suspicion is raised.

[00177] A biomarker that can specifically identify patients as higher/high risk of aortic dilatation can be used in certain embodiments to risk stratify patients into those who require more definitive invasive/higher risk investigations and those requiring routine monitoring only.

[00178] In certain embodiments, the subject has an increased risk or likelihood of suffering from, or being susceptible to, aortic root dilatation.

[00179] In certain embodiments, the method comprises identifying an increased level of FSLT-3 and one or more clinical features, such as the presence and/or absence of aortic root dilatation, and/or clinical features associated with the progression of aortic root dilatation. [00180] In certain embodiments, the clinical features comprise clinical features associated with the presence and/or absence of aortic root dilatation, and/or clinical features associated with the progression of aortic root dilatation. Clinical features are known in the art.

[00181] In certain embodiments, a plasma level of folli statin-like 3 greater than 9500 pg/ml is indicative that the subject is suffering from, or susceptible to, aortic root dilatation.

[00182] In certain embodiments, a plasma level of folli statin-like 3 greater than 9000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, aortic root dilatation.

[00183] In certain embodiments, a plasma level of follistatin-3 in the range from 9500 to 38000 pg/ml is indicative that the subject is suffering from, or susceptible to, sortie root dilatation. Other ranges are contemplated.

[00184] In certain embodiments, a plasma level of follistatin-3 in the range from 9000 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, aortic root dilatation. Other ranges are contemplated.

[00185] In certain embodiments, the method comprises determining the level of one or more other markers.

[00186] In certain embodiments, the method comprises determining the level of galectin-3. In certain embodiments, the method comprises determining the level of FSTL-3 and galectin-3.

[00187] In certain embodiments, the method comprises identifying an increased level of galectin-3 in the subject.

[00188] In certain embodiments, the condition associated with cardiac fibrotic remodelling is chemotherapy-induced cardiovascular toxicity. [00189] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to, chemotherapy-induced cardiovascular toxicity, the method comprising identifying an increased level of folli statin-like 3 in the subject.

[00190] Chemotherapy for many cancers does not come without risk and can result in treatment-related toxicities that produce serious side effects. Chemotherapy-induced cardiotoxicity (CIC) is a poisonous or detrimental effect upon the heart that can delay cancer treatment, decrease survival, and increase morbidity.

[00191] CIC can affect anywhere between 5 and 25% of patients receiving chemotherapy, depending on the specific agent and the total dose. Currently, the diagnosis of CIC is made either when a patient presents with symptoms of heart disease, or during monitoring with ultrasound or MRI scans of the heart, both of which are costly. By that stage there are usually significant changes in heart structure and function that require urgent intensive long-term treatment and complete recovery rate is low.

[00192] Early treatment with conventional heart failure medications significantly improves recovery from CIC and leads to much better long term prognosis and survival. At present there are no reliable ways to predict which patients are more likely to develop CIC before initiation of chemotherapy.

[00193] The identification of a biomarker prior to initiation of chemotherapy can be used to identify those patients who would develop CIC. These patients may be more intensively monitored and receive heart-specific treatment early to avoid detrimental effect on their heart health, quality of life and survival.

[00194] In certain embodiments, the subject has an increased risk or likelihood of suffering from, or being susceptible to, chemotherapy -induced cardiovascular toxicity.

[00195] In certain embodiments, the method comprises identifying an increased level of FSLT-3 and one or more clinical features.

[00196] In certain embodiments, the clinical features comprise clinical features associated with the presence and/or absence of chemotherapy-induced cardiovascular toxicity, and/or clinical features associated with the progression of chemotherapy- induced cardiovascular toxicity. Clinical features are known in the art, such as development of hypertension, heart failure, arrhythmias.

[00197] In certain embodiments, a plasma level of folli statin-like 3 greater than 6500 pg/ml is indicative that the subject is suffering from, or susceptible to, chemotherapy- induced cardiovascular toxicity.

[00198] In certain embodiments, a plasma level of folli statin-like 3 greater than 6500 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, chemotherapy-induced cardiovascular toxicity.

[00199] In certain embodiments, a plasma level of follistatin-3 in the range from 6500 to 38000 pg/ml is indicative that the subject is suffering from, or susceptible to, chemotherapy-induced cardiovascular toxicity. In certain embodiments, a plasma level of follistatin-3 in the range from 8000 to 38000 pg/ml is indicative that the subject is suffering from, or susceptible to, chemotherapy-induced cardiovascular toxicity. In certain embodiments, a plasma level of follistatin-3 in the range from 8200 to 38000 pg/ml is indicative that the subject is suffering from, or susceptible to, chemotherapy- induced cardiovascular toxicity. Other ranges are contemplated.

[00200] In certain embodiments, a plasma level of follistatin-3 in the range from 6500 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, chemotherapy-induced cardiovascular toxicity. In certain embodiments, a plasma level of follistatin-3 in the range from 8000 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, chemotherapy-induced cardiovascular toxicity. In certain embodiments, a plasma level of follistatin-3 in the range from 8200 to 38000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, chemotherapy-induced cardiovascular toxicity. Other ranges are contemplated.

[00201] In certain embodiments, the method comprises determining the level of one or more other markers, such as Troponin, NT-proB P, and sST-2. Methods for determining the level of such markers are known in the art.

[00202] In certain embodiments, the method comprises determining the level of galectin-3. In certain embodiments, the method comprises determining the level of FSTL-3 and galectin-3.

[00203] In certain embodiments, the method comprises identifying an increased level of galectin-3 in the subject.

[00204] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling, the method comprising:

detecting follistatin-like 3 from the subject; and

identifying the subject as suffering from, or being susceptible to, a cardiac condition associated with cardiac fibrotic remodelling on the basis of the level of the follistatin-like 3 detected.

[00205] In certain embodiments, the methods as described herein comprise using a computer processor means to process data associated with the level of the follistatin-like 3. Use of a computer processor means to process data are known in the art, utilising suitable algorithms.

[00206] In certain embodiments, the method comprises using a computer processor means to process data associated with the level of the follistatin-like 3 to generate a likelihood and/or risk of a cardiac condition associated with cardiac fibrotic remodelling occurring in the subject.

[00207] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling, the method comprising:

detecting follistatin-like 3 from the subject;

using a computer processor means to process data associated with the level of the follistatin-like 3 to generate a likelihood and/or risk of a cardiac condition associated with cardiac fibrotic remodelling occurring in the subject; and identifying the presence or absence of the condition in the subject on the basis of the likelihood and/or risk generated.

[00208] Certain embodiments of the present disclosure provide a method of screening for a condition associated with cardiac fibrotic remodelling occurring in a subject.

[00209] Certain embodiments of the present disclosure provide a method of screening for a condition associated with cardiac fibrotic remodelling in a subject, the method comprising determining the level of follistatin-like 3 in the subject. Levels and ranges of FSTL-3 associated with the conditions are as described herein.

[00210] In certain embodiments, the level of the follistatin-like 3 so determined is indicative of whether or not the subject is suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling.

[00211] In certain embodiments, the level of the follistatin-like 3 so determined is indicative of the risk or likelihood of the suffering from, or susceptible to, the condition associated with cardiac fibrotic remodelling.

[00212] In certain embodiments, an increased level of follastatin-like 3 is indicative that the subject is suffering from, or susceptible to, the condition associated with cardiac fibrotic remodelling.

[00213] In certain embodiments, an increased level of follastatin-like 3 is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, the condition associated with cardiac fibrotic remodelling.

[00214] Levels of follastatin-3 indicative of a condition associated with cardiac fibrotic remodelling are as described herein, and may be used to screen for the presence or absence, or likelikood or risk, of a condition associated with cardiac fibrotic remodelling in the subject.

[00215] Subjects are as described herein. Conditions associated with cardiac fibrotic remodelling are as described herein. [00216] In certain embodiments, the subject does not show any increase in the level of troponin and/or BNP at the time of determining the level of follistatin-like 3.

[00217] Methods for detecting FSTL-3 and determining the level of FSTL-3 are as described herein. In certain embodiments, the method comprises determining the level of FSTL-3 by an immunological method.

[00218] In certain embodiments, the level of FSTL-3 is compared to a level of the marker that is present in a subject not suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling. In certain embodiments, the level of FSTL- 3 is compared to a level of the marker that is a level of the marker in the general population. In certain embodiments, the level of FSTL-3 is compared to a level of the marker that is indicative of a subject not suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling. In certain embodiments, the level of FSTL- 3 is compared to a level of the marker that is indicative of a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling.

[00219] In certain embodiments, the method comprises detecting FSTL-3 and one or more other markers.

[00220] In certain embodiments, the method comprises determining the level of FSTL- 3 and one or more other markers, such as troponin and/or BNP.

[00221] In certain embodiments, the one or more other markers is galectin-3. Methods for detecting galectin-3 and determining the level of galectin-3 are known in the art. In certain embodiments, the method comprises determining the level of galectin-3.

[00222] In certain embodiments, the method comprises determining the level of FSTL- 3 and galectin-3.

[00223] In certain embodiments, the method comprises identifying an increased level of galectin-3 in the subject.

[00224] In certain embodiments, the method comprises determining the level of galectin-3 by an immunological method. [00225] In certain embodiments, the level of galectin-3 is compared to a level of the marker that is present in a subject not suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling. In certain embodiments, the level of galectin-3 is compared to a level of the marker that is a level of the marker in the general population. In certain embodiments, the level of galectin-3 is compared to a level of the marker that is indicative of a subject not suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling. In certain embodiments, the level of galectin-3 is compared to a level of the marker that is indicative of a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling.

[00226] In certain embodiments, the method comprises obtaining a biological sample from the subject. Biological samples are as described herein.

[00227] In certain embodiments, the biological sample comprises one or more of blood, plasma and serum. In certain embodiments, the FSTL-3 is a blood marker, a plasma marker and/or a serum marker.

[00228] In certain embodiments, the method comprises processing the biological sample to allow detection of markers. In certain embodiments, the method comprises processing a biological sample obtained from the subject and detecting the markers. In certain embodiments, the method comprises processing the biological sample to allow detection of markers in the biological sample.

[00229] In certain embodiments, the method comprises obtaining a biological sample from the subject and processing the sample to allow detection of the folli statin-like 3 and/or one or more other markers, such as galectin-3.

[00230] In certain embodiments, the method comprises determining the level of FSLT- 3 and assessing one or more clinical features. Clinical features are as described herein.

[00231] Conditions associated with cardiac fibrotic remodelling are as described herein. In certain embodiments, the condition associated with cardiac fibrotic remodelling is selected from one of cardiac hypertrophy, heart failure, response to cardiac resynchronisation therapy, paroxysmal atrial fibrillation, aortic root dilatation with aortic valve disease, chemotherapy-induced cardiovascular toxicity and stroke arising from atrial fibrillation.

[00232] In certain embodiments, the condition associated with cardiac fibrotic remodelling is cardiac hypertrophy.

[00233] Certain embodiments of the present disclosure provide a method of screening for a condition associated with cardiac hypertrophy in a subject, the method comprising determining the level of folli statin-like 3 in the subject.

[00234] In certain embodiments, the method comprises determining the level of FSLT- 3 and assessing one or more clinical features associated with cardiac hypertrophy. Clinical features associated with cardiac hypertrophy are as described herein.

[00235] In certain embodiments, a plasma level of folli statin-like 3 greater than 6500 pg/ml is indicative that the subject is suffering from, or susceptible to, cardiac hypertrophy.

[00236] In certain embodiments, a plasma level of folli statin-like 3 greater than 5500 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, cardiac hypertrophy.

[00237] In certain embodiments, the method comprises determining the level of one or more other markers.

[00238] In certain embodiments, the method comprises determining the level of galectin-3. In certain embodiments, the method comprises identifying an increased level of galectin-3

[00239] In certain embodiments, the method comprises determining the level of FSTL- 3 and galectin-3.

[00240] In certain embodiments, an increased level of FSTL-3 and galectin-3 is indicative that the subject has an increased risk or likelihood of suffering from, or is susceptible to, cardiac hypertrophy. [00241] In certain embodiments, the method comprises determining the level of one or more of N-terminal pro-brain natriuretic peptide, mid-regional pro-atrial natriuretic peptide, mid-regional pro-adrenomedullin, C-terminal pro-endothelin and osteoprotegerin.

[00242] In certain embodiments, an increased level of FSLT-3 in combination with one or more clinical features is indicative that the subject is suffering from, or susceptible to, cardiac hypertrophy.

[00243] In certain embodiments, an increased level of FSLT-3 in combination with one or more clinical features is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, cardiac hypertrophy.

[00244] In certain embodiments, the condition associated with cardiac fibrotic remodelling is heart failure.

[00245] Certain embodiments of the present disclosure provide a method of screening for heart failure in a subject, the method comprising determining the level of follistatin- like 3 in the subject.

[00246] In certain embodiments, the heart failure is acute heart failure. In certain embodiments, the heart failure is chronic heart failure.

[00247] In certain embodiments, the subject has an increased risk or likelihood of suffering from, or being susceptible to, heart failure.

[00248] In certain embodiments, the subject has an increased risk or likelihood of suffering from, or being susceptible to, acute heart failure.

[00249] In certain embodiments, the subject has an increased risk or likelihood of suffering from, or being susceptible to, chronic heart failure.

[00250] In certain embodiments, the method comprises determining the level of FSLT- 3 and assessing one or more clinical features associated with heart failure. Clinical features associated with heart failure are as described herein. [00251] In certain embodiments, a plasma level of folli statin-like 3 greater than 7000 pg/ml is indicative that the subject is suffering from, or susceptible to, heart failure.

[00252] In certain embodiments, a plasma level of folli statin-like 3 greater than 6500 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or is susceptible to, heart failure.

[00253] In certain embodiments, the method comprises determining the level of one or more other markers.

[00254] In certain embodiments, the method comprises determining the level of galectin-3. In certain embodiments, the method comprises identifying an increased level of galectin-3.

[00255] In certain embodiments, the method comprises determining the level of FSTL- 3 and galectin-3.

[00256] In certain embodiments, an increased level of FSTL-3 and galectin-3 is indicative that the subject has an increased risk or likelihood of suffering from, or is susceptible to, heart failure.

[00257] In certain embodiments, the method comprises determining the level of one or more of B P and NT-proBNP, MR-proA P, MR-proADM, troponins, sST2, and GDF- 15. Methods for determining the levels of such markers are known in the art.

[00258] In certain embodiments, an increased level of FSLT-3 in combination with one or more clinical features is indicative that the subject is suffering from, or susceptible to, heart failure.

[00259] In certain embodiments, an increased level of FSLT-3 in combination with one or more clinical features is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, heart failure.

[00260] In certain embodiments, a plasma level of folli statin-like 3 greater than 10000 pg/ml is indicative that the subject is suffering from, or susceptible to, acute heart failure.

[00261] In certain embodiments, a plasma level of folli statin-like 3 greater than 9000 pg/ml is indicative that the subject has an increased risk or likelihood that the subject is suffering from, or susceptible to, acute heart failure.

[00262] In certain embodiments, a plasma level of folli statin-like 3 greater than 7000 pg/ml is indicative that the subject is suffering from, or susceptible to, chronic heart failure.

[00263] In certain embodiments, a plasma level of follistatin-like 3 greater than 6500 pg/ml is indicative that the subject has increased risk or likelihood of suffering from, or being susceptible to, chronic heart failure.

[00264] In certain embodiments, the condition associated with cardiac fibrotic remodelling is response to cardiac resynchronisation therapy.

[00265] Certain embodiments of the present disclosure provide a method of screening for response to cardiac resynchronisation therapy in a subject, the method comprising determining the level of follistatin-like 3 in the subject.

[00266] In certain embodiments, the method comprises determining the level of FSLT- 3 and assessing one or more clinical features associated with heart failure. Clinical features associated with heart failure are as described herein.

[00267] In certain embodiments, a plasma level of follistatin-like 3 less than 13000 pg/ml is indicative that the subject will be responsive to cardiac resynchronisation therapy.

[00268] In certain embodiments, a plasma level of follistatin-like 3 greater than 10000 pg/ml is indicative that the subject has an increased likelihood of being responsive to cardiac resynchronisation therapy.

[00269] In certain embodiments, the method comprises determining the level of one or more other markers. [00270] In certain embodiments, the method comprises determining the level of galectin-3. In certain embodiments, the method comprises identifying an increased level of galectin-3.

[00271] In certain embodiments, the method comprises determining the level of FSTL- 3 and galectin-3.

[00272] In certain embodiments, an increased level of FSTL-3 and galectin-3 is indicative that the subject will be responsive to cardiac resynchronisation therapy.

[00273] In certain embodiments, an increased level of FSTL-3 and galectin-3 is indicative that the subject has an increased likelihood of being responsive to cardiac resynchronisation therapy.

[00274] In certain embodiments, the method comprises determining the level of one or more of B P and NT-proBNP, MR-proA P, MR-proADM, troponins, sST2, and GDF- 15. Methods for determining the levels of such markers are known in the art.

[00275] In certain embodiments, an increased level of FSLT-3 in combination with one or more clinical features is indicative that the subject will be responsive to cardiac resynchronisation therapy.

[00276] In certain embodiments, an increased level of FSLT-3 in combination with one or more clinical features is indicative that the subject will be have an increased likelihood of being responsive to cardiac resynchronisation therapy.

[00277] In certain embodiments, a plasma level of galectin-3 of greater than 15 ng/ml is indicative that the subject has a decreased likelihood of being responsive to cardiac resynchronisation therapy, and a plasma level of galectin-3 of less than 12.5 ng/ml is indicative that the subject has an increased likelihood of being responsive to cardiac resynchronisation therapy.

[00278] In certain embodiments, the condition associated with cardiac fibrotic remodelling is paroxysmal atrial fibrillation and/or stroke arising from atrial fibrillation. [00279] Certain embodiments of the present disclosure provide a method of screening for paroxysmal atrial fibrillation a subject, the method comprising determining the level of folli statin-like 3 in the subject.

[00280] In certain embodiments, the method comprises determining the level of FSLT- 3 and assessing one or more clinical features associated with atrial fibrillation. Clinical features associated with atrial fibrillation are as described herein.

[00281] In certain embodiments, a plasma level of folli statin-like 3 greater than 9000 pg/ml is indicative that the subject is suffering from, or susceptible to, paroxysmal atrial fibrillation.

[00282] In certain embodiments, a plasma level of folli statin-like 3 greater than 6600 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, paroxysmal atrial fibrillation.

[00283] In certain embodiments, the method comprises determining the level of one or more other markers.

[00284] In certain embodiments, the method comprises determining the level of galectin-3. In certain embodiments, the method comprises identifying an increased level of galectin-3.

[00285] In certain embodiments, the method comprises determining the level of FSTL- 3 and galectin-3.

[00286] In certain embodiments, an increased level of FSTL-3 and galectin-3 is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, paroxysmal atrial fibrillation.

[00287] In certain embodiments, the method comprises determining the level of one or more of BNP, NT-proBNP and a troponin. Methods for determining the levels of such markers are known in the art.

[00288] In certain embodiments, an increased level of FSLT-3 in combination with one or more clinical features is indicative that the subject is suffering from, or susceptible to, paroxysmal atrial fibrillation.

[00289] In certain embodiments, an increased level of FSLT-3 in combination with one or more clinical features is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, paroxysmal atrial fibrillation.

[00290] Certain embodiments of the present disclosure provide a method of screening for susceptibility of a subject to stroke arising from atrial fibrillation, the method comprising determining the level of folli statin-like 3 in the subject.

[00291] In certain embodiments, the method comprises determining the level of FSLT- 3 and assessing one or more clinical features associated with atrial fibrillation. Clinical features associated with atrial fibrillation are as described herein.

[00292] In certain embodiments, a plasma level of folli statin-like 3 greater than 9600 pg/ml is indicative that the subject is susceptible to stroke arising from atrial fibrillation.

[00293] In certain embodiments, a plasma level of folli statin-like 3 greater than 8600 pg/ml is indicative that the subject has an increased risk or likelihood of susceptibility to stroke arising from atrial fibrillation.

[00294] In certain embodiments, the method comprises determining the level of one or more other markers.

[00295] In certain embodiments, the method comprises determining the level of galectin-3. In certain embodiments, the method comprises identifying an increased level of galectin-3.

[00296] In certain embodiments, the method comprises determining the level of FSTL- 3 and galectin-3.

[00297] In certain embodiments, an increased level of FSTL-3 and galectin-3 is indicative that the subject has an increased risk or likelihood of susceptibility to stroke or stroke arising from atrial fibrillation. [00298] In certain embodiments, the method comprises determining the level of one or more of BNP, NT-proBNP and a troponin. Methods for determining the levels of such markers are known in the art.

[00299] In certain embodiments, an increased level of FSLT-3 in combination with one or more clinical features is indicative that the subject is susceptible to stroke or stroke arising from atrial fibrillation cardiac hypertrophy.

[00300] In certain embodiments, an increased level of FSLT-3 in combination with one or more clinical features is indicative that the subject has an increased risk or likelihood of suffering from a stroke arising from atrial fibrillation.

[00301] In certain embodiments, the condition associated with cardiac fibrotic remodelling is aortic root dilatation.

[00302] Certain embodiments of the present disclosure provide a method of screening for aortic root dilatation in a subject, the method comprising determining the level of folli statin-like 3 in the subject.

[00303] In certain embodiments, the method comprises determining the level of FSLT- 3 and assessing one or more clinical features associated with aortic root dilatation. Clinical features associated with cardiac hypertrophy are as described herein.

[00304] In certain embodiments, a plasma level of folli statin-like 3 greater than 9500 pg/ml is indicative that the subject is suffering from, or susceptible to, aortic root dilatation.

[00305] In certain embodiments, a plasma level of folli statin-like 3 greater than 9000 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or is susceptible to, aortic root dilatation.

[00306] In certain embodiments, the method comprises determining the level of one or more other markers.

[00307] In certain embodiments, the method comprises determining the level of galectin-3. In certain embodiments, the method comprises identifying an increased level of galectin-3.

[00308] In certain embodiments, the method comprises determining the level of FSTL- 3 and galectin-3.

[00309] In certain embodiments, an increased level of FSTL-3 and galectin-3 is indicative that the subject has an increased risk or likelihood of suffering from, or is susceptible to, aortic root dilatation.

[00310] In certain embodiments, an increased level of FSLT-3 in combination with one or more clinical features is indicative that the subject is suffering from, or susceptible to, aortic root dilatation.

[00311] In certain embodiments, an increased level of FSLT-3 in combination with one or more clinical features is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, aortic root dilatation.

[00312] In certain embodiments, the condition associated with cardiac fibrotic remodelling is chemotherapy-induced cardiovascular toxicity.

[00313] Certain embodiments of the present disclosure provide a method of screening for chemotherapy-induced cardiovascular toxicity the method comprising determining the level of folli statin-like 3 in the subject.

[00314] In certain embodiments, the method comprises determining the level of FSLT- 3 and assessing one or more clinical features associated with chemotherapy-induced cardiovascular toxicity. Clinical features associated with chemotherapy-induced cardiovascular toxicity are as described herein.

[00315] In certain embodiments, a plasma level of folli statin-like 3 greater than 6500 pg/ml is indicative that the subject is suffering from, or susceptible to, chemotherapy- induced cardiovascular toxicity.

[00316] In certain embodiments, a plasma level of folli statin-like 3 greater than 6500 pg/ml is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, chemotherapy-induced cardiovascular toxicity.

[00317] In certain embodiments, the method comprises determining the level of one or more other markers.

[00318] In certain embodiments, the method comprises determining the level of galectin-3. In certain embodiments, the method comprises identifying an increased level of galectin-3.

[00319] In certain embodiments, the method comprises determining the level of FSTL- 3 and galectin-3.

[00320] In certain embodiments, an increased level of FSTL-3 and galectin-3 is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, chemotherapy-induced cardiovascular toxicity.

[00321] In certain embodiments, an increased level of FSLT-3 in combination with one or more clinical features is indicative that the subject is suffering from, or susceptible to, chemotherapy-induced cardiovascular toxicity.

[00322] In certain embodiments, an increased level of FSLT-3 in combination with one or more clinical features is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, chemotherapy-induced cardiovascular toxicity.

[00323] Certain embodiments of the present disclosure provide a method of screening for a condition associated with cardiac fibrotic remodelling in a subject, the method comprising determining the level of follistatin-like 3 in the subject, wherein the level of the follistatin-like 3 so determined is indicative of whether or not the subject is suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling.

[00324] Certain embodiments of the present disclosure provide a method of screening for a condition associated with cardiac fibrotic remodelling in a subject, the method comprising determining the level of follistatin-like 3 in the subject, wherein the level of the follistatin-like 3 so determined is indicative of the risk or likelihood of the suffering from, or being susceptible to, the condition associated with cardiac fibrotic remodelling.

[00325] Certain embodiments of the present disclosure provide a method of screening for a condition associated with cardiac fibrotic remodelling in a subject, the method comprising determining the level of follistatin-like 3 in the subject, wherein an increased level of follistatin-like 3 is indicative that the subject is suffering from, or susceptible to, the condition associated with cardiac fibrotic remodelling.

[00326] Certain embodiments of the present disclosure provide a method of screening for a condition associated with cardiac fibrotic remodelling occurring in a subject, the method comprising determining the level of follistatin-like 3 in the subject, wherein an increased level of follastatin-like 3 is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, the condition associated with cardiac fibrotic remodelling.

[00327] Certain embodiments of the present disclosure provide a method of determining the likelihood or risk of a condition associated with cardiac fibrotic remodelling occurring in a subject.

[00328] Certain embodiments of the present disclosure provide a method of determining the likelihood or risk of a condition associated with cardiac fibrotic remodelling in a subject, the method comprising determining the level of FSTL-3 in the subject and determining the likelihood or risk of a condition associated with cardiac fibrotic remodelling occurring in a subject on the basis of the level of FSTL-3 so determined.

[00329] In certain embodiments, an increased level of follastatin-like 3 is indicative that the subject has an increased risk or likelihood of suffering from, or being susceptible to, the condition associated with cardiac fibrotic remodelling.

[00330] Subjects are as described herein. In certain embodiments, the subject has an increased risk or likelihood of suffering from, or being susceptible to, a condition associated with cardiac fibrotic remodelling. [00331] Methods for detecting FSTL-3 and determining the level of FSTL-3 are as described herein. In certain embodiments, the method comprises determining the level of FSTL-3 by an immunological method.

[00332] In certain embodiments, the level of FSTL-3 is compared to a level of the marker that is present in a subject not suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling. In certain embodiments, the level of FSTL- 3 is compared to a level of the marker that is a level of the marker in the general population. In certain embodiments, the level of FSTL-3 is compared to a level of the marker that is indicative of a subject not suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling. In certain embodiments, the level of FSTL- 3 is compared to a level of the marker that is indicative of a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling.

[00333] In certain embodiments, the method comprises detecting FSTL-3 and one or more other markers.

[00334] In certain embodiments, the method comprises determining the level of FSTL- 3 and one or more other markers, such as a troponin and/or B P.

[00335] In certain embodiments, the one or more other markers is galectin-3. Methods for detecting galectin-3 and determining the level of galectin-3 are known in the art. In certain embodiments, the method comprises identifying an increased level of galectin-3.

[00336] In certain embodiments, the method comprises determining the level of galectin-3 by an immunological method.

[00337] In certain embodiments, the method comprises determining the level of FSTL- 3 and galectin-3.

[00338] In certain embodiments, the method comprises identifying an increased level of FSTL-3 and galectin-3.

[00339] In certain embodiments, the level of galectin-3 is compared to a level of the marker that is present in a subject not suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling. In certain embodiments, the level of galectin-3 is compared to a level of the marker that is a level of the marker in the general population. In certain embodiments, the level of galectin-3 is compared to a level of the marker that is indicative of a subject not suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling. In certain embodiments, the level of galectin-3 is compared to a level of the marker that is indicative of a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling.

[00340] In certain embodiments, the method comprises obtaining a biological sample from the subject. Biological samples are as described herein.

[00341] In certain embodiments, the biological sample comprises one or more of blood, plasma and serum. In certain embodiments, the FSTL-3 is a blood marker, a plasma marker and/or a serum marker.

[00342] In certain embodiments, the method comprises processing the biological sample to allow detection of markers. In certain embodiments, the method comprises processing a biological sample obtained from the subject and detecting the markers. In certain embodiments, the method comprises processing the biological sample to allow detection markers in the biological sample.

[00343] In certain embodiments, the method comprises obtaining a biological sample from the subject and processing the sample to allow detection of the folli statin-like 3 and/or one or more other markers.

[00344] In certain embodiments, the method comprises determining the level of FSLT- 3 and assessing one or more clinical features. Clinical features are as described herein.

[00345] Conditions associated with cardiac fibrotic remodelling are as described herein.

[00346] In certain embodiments, the condition associated with cardiac fibrotic remodelling is selected from one of cardiac hypertrophy, heart failure, response to cardiac resynchronisation therapy, paroxysmal atrial fibrillation, a stroke arising from atrial fibrillation, aortic root dilatation with aortic valve disease, chemotherapy-induced cardiovascular toxicity and stroke arising from atrial fibrillation. [00347] In certain embodiments, the methods as described herein are used for the diagnosis or prognosis of a condition associated with cardiac fibrotic remodelling in a subject.

[00348] In certain embodiments, the condition associated with cardiac fibrotic remodelling is cardiac hypertrophy.

[00349] Certain embodiments of the present disclosure provide a method of determining the likelihood or risk of cardiac hypertrophy in a subject, the method comprising determining the level of FSTL-3 in the subject and determining the likelihood or risk of cardiac hypertrophy occurring in a subject on the basis of the level of FSTL-3 so determined.

[00350] In certain embodiments, an increased level of FSTL-3 is indicative that the subject has an increased or likelihood of suffering from, or being susceptible to cardiac hypertrophy.

[00351] In certain embodiments, the condition associated with cardiac fibrotic remodelling is heart failure.

[00352] Certain embodiments of the present disclosure provide a method of determining the likelihood or risk of heart failure in a subject, the method comprising determining the level of FSTL-3 in the subject and determining the likelihood or risk of heart failure occurring in a subject on the basis of the level of FSTL-3 so determined.

[00353] In certain embodiments, an increased level of FSTL-3 is indicative that the subject has an increased or likelihood of suffering from, or being susceptible to heart failure.

[00354] In certain embodiments, the condition associated with cardiac fibrotic remodelling is response to cardiac resynchronisation therapy.

[00355] Certain embodiments of the present disclosure provide a method of determining the likelihood of a subject being responsive to cardiac resynchronisation therapy, the method comprising determining the level of FSTL-3 in the subject and determining the likelihood of the subject being responsive to cardiac resynchronisation therapy on the basis of the level of FSTL-3 so determined.

[00356] In certain embodiments, the method is used to determine the responsiveness of a subject with chronic heart failure to cardiac resynchronisation therapy. In certain embodiments, an increased level of FSTL-3 is indicative that the subject is likely to be responsive to CRT or has an increased likelihood of being responsive to CRT. Methods for determining the responsiveness to CRT are as described herein.

[00357] Certain embodiments of the present disclosure provide a method of determining the responsiveness of a subject with chronic heart failure to cardiac resynchronisation therapy, the method comprising determining the level of follistatin- like 3 in the subject and determining the responsiveness of the subject to cardiac resynchronisation therapy.

[00358] In certain embodiments, the condition associated with cardiac fibrotic remodelling is paroxysmal atrial fibrillation.

[00359] Certain embodiments of the present disclosure provide a method of determining the likelihood or risk of paroxysmal atrial fibrillation occurring in a subject, the method comprising determining the level of FSTL-3 in the subject and determining the likelihood or risk of paroxysmal atrial fibrillation occurring in a subject on the basis of the level of FSTL-3 so determined.

[00360] In certain embodiments, an increased level of FSTL-3 is indicative that the subject has an increased or likelihood of suffering from, or being susceptible to paroxysmal atrial fibrillation.

[00361] In certain embodiments, the condition associated with cardiac fibrotic remodelling is stroke arising from atrial fibrillation.

[00362] Certain embodiments of the present disclosure provide a method of determining the likelihood or risk of a stroke arising from atrial fibrillation occurring in a subject, the method comprising determining the level of FSTL-3 in the subject and determining the likelihood or risk of a stroke arising from atrial fibrillation occurring in a subject on the basis of the level of FSTL-3 so determined.

[00363] In certain embodiments, an increased level of FSTL-3 is indicative that the subject has an increased or likelihood of suffering from, or being susceptible to, a stroke arising from atrial fibrillation.

[00364] In certain embodiments, the condition associated with cardiac fibrotic remodelling is aortic root dilatation with aortic valve disease.

[00365] Certain embodiments of the present disclosure provide a method of determining the likelihood or risk of aortic root dilatation with aortic valve disease occurring in a subject, the method comprising determining the level of FSTL-3 in the subject and determining the likelihood or risk of aortic root dilatation with aortic valve disease occurring in a subject on the basis of the level of FSTL-3 so determined.

[00366] In certain embodiments, an increased level of FSTL-3 is indicative that the subject has an increased or likelihood of suffering from, or being susceptible, aortic root dilatation.

[00367] In certain embodiments, the condition associated with cardiac fibrotic remodelling is chemotherapy-induced cardiovascular toxicity.

[00368] Certain embodiments of the present disclosure provide a method of determining the likelihood of chemotherapy-induced cardiovascular toxicity occurring in a subject, the method comprising determining the level of FSTL-3 in the subject and determining the likelihood of chemotherapy-induced cardiovascular toxicity occurring in the subject on the basis of the level of FSTL-3 so determined.

[00369] In certain embodiments, an increased level of FSTL-3 is indicative that the subject has an increased or likelihood of suffering from, or being susceptible to chemotherapy-induced cardiovascular toxicity.

[00370] Certain embodiments of the present disclosure provide a method of determining the likelihood or risk of a condition associated with cardiac fibrotic remodelling occurring in a subject, the method comprising: determining the level of follistatin-like 3 from the subject; and

[00371] Certain embodiments of the present disclosure provide a method of monitoring progression of a condition associated with cardiac fibrotic remodelling occurring in a subject.

[00372] Certain embodiments of the present disclosure provide a method of monitoring progression of a condition associated with cardiac fibrotic remodelling occurring in a subject, the method comprising determining the level of follistatin-like 3 in the subject and determining the extent of disease progression on the basis of the level of follistatin- like 3 determined.

[00373] In certain embodiments, an increased level of follistatin-like 3 is indicative of continued or increasing progression of the condition.

[00374] In certain embodiments, the methods as described use a computer processor means to process data associated with the level of the follistatin-like 3 and/or one or more other markers to generate a likelihood and/or risk of the presence or absence of the condition in the subject.

[00375] In certain embodiments, the methods comprises using a computer processor means to process data associated with the level of FSTL-3 and/or one or more other markers to generate or determine a likelihood and/or risk of condition, and/or determine the presence or absence of the condition. Methods of determining likelihood and/or risk are contemplated.

[00376] In certain embodiments, the method comprises transferring the data/information over the internet to a computer processing means.

[00377] In certain embodiments, the method comprises transferring data associated with the one or more characteristics over the internet to a computer processing means to generate a likelihood or risk.

[00378] For example, web-based statistical software can be used to assesses the risk and provide a probability or risk or likelihood.

[00379] In certain embodiments, the methods comprise processing the data/information to classify the risk in the subject as having increased risk, a high risk, a moderate risk, a low risk, a normal risk or a decreased risk. In certain embodiments, the method comprises processing the data/information to identity the presence or absence of a condition. In certain embodiments, the method comprises processing the data/information to exclude the presence of a condition.

[00380] Computer processing means are known in the art. Method for sending and/or receiving data/information are known in the art.

[00381] Certain embodiments of the present disclosure provide a system for determining the likelihood or risk using a computer processor. Systems utilising computer processors are known in the art. Examples are as described herein.

[00382] Certain embodiments of the present disclosure provide a system for determining the risk using a computer processor configured to process a method as described herein.

[00383] Certain embodiments of the present disclosure provide a computer readable medium.

[00384] Certain embodiments of the present disclosure provide a computer-readable medium encoded with programming instructions executable by a computer processor means to allow the computer processor means to receive data associated with the presence and/or level of FSTL-3 and process the data to generate a likelihood and/or risk.

[00385] Certain embodiments of the present disclosure provide a computer processor means comprising a computer-readable medium as described herein. [00386] Certain embodiments of the present disclosure provide method of preventing and/or treating a condition associated with cardiac fibrotic remodelling in a subject.

[00387] Certain embodiments of the present disclosure provide method of preventing and/or treating a condition associated with cardiac fibrotic remodelling in a subject, the method comprising determining the likelihood or risk of a condition associated with cardiac fibrotic remodelling occurring in a subject on the basis of the level of follistatin- like 3 in the subject, and treating the subject on the basis of the level of follistatin-like 3 so determined.

[00388] Certain embodiments of the present disclosure provide method of preventing and/or treating a condition associated with cardiac fibrotic remodelling in a subject, the method comprising:

determining the level of follistatin-like 3 in the subject; and

treating the subject on the basis of the level of follistatin-like 3 so determined.

[00389] Certain embodiments of the present disclosure provide method of preventing and/or treating a condition associated with cardiac fibrotic remodelling in a subject, the method comprising:

determining the level of follistatin-like 3 in the subject; and

treating the subject on the basis of one or more clinical features of the subject and the level of follistatin-like 3 so determined.

[00390] Certain embodiments of the present disclosure provide a method of preventing and/or treating a condition associated with cardiac fibrotic remodelling in a subject, the method comprising using a method as described herein to determine the likelihood or risk of the condition occurring and treating the subject on the basis of the likelihood or risk so determined.

[00391] Certain embodiments of the present disclosure provide a method of preventing and/or treating a condition associated with cardiac fibrotic remodelling in a subject by diagnosing the presence or likelihood of the condition occurring as described herein, and treating/administering the subject with a suitable agent.

[00392] Treatments for a condition associated with cardiac fibrotic remodelling are known in the art. For example, the diagnosis and management of cardiac hypertrophy is as described in Elliot et al. (2014) "2014 ESC Guidelines on diagnosis and management of hypertrophic cardiomyopathy" European Heart Journal (2014) 35, 2733-2779; the diagnosis and treatment of acute and chronic heart failure is as described in Ponikowski et al. (2016) "2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure" European Heart Journal (2016) doi: 10.1093/eurheartj/ehwl28; the diagnosis and treatment of acute and chronic heart failure is as described in Piepolu et al. (2016) "2016 European Guidelines on cardiovascular disease prevention in clinical practice" European Heart Journal doi : 10.1093/eurheartj/ehwl 06.

[00393] In certain embodiments, the condition associated with cardiac fibrotic remodelling is selected from one of cardiac hypertrophy, heart failure, response to cardiac resynchronisation therapy, paroxysmal atrial fibrillation, aortic root dilatation with aortic valve disease, chemotherapy-induced cardiovascular toxicity and stroke arising from atrial fibrillation.

[00394] Certain embodiments of the present disclosure provide a method of identifying a subject suitable for treatment for a condition associated with cardiac fibrotic remodelling.

[00395] Certain embodiments of the present disclosure provide a method of identifying a subject suitable for treatment for a condition associated with cardiac fibrotic remodelling in the subject, the method comprising:

determining the level of folli statin-like 3 in the subject; and

identifying the subject as a subject suitable for treatment on the basis of the level of follistatin-like 3 so determined.

[00396] Certain embodiments of the present disclosure provide a method of identifying a subject suitable for treatment for a condition associated with cardiac fibrotic remodelling in the subject, the method comprising:

determining the level of follistatin-like 3 in the subject; and identifying the subject as a subject suitable for treatment on the basis of one or more clinical features of the subject and the level of folli statin-like 3 so determined.

[00397] In certain embodiments, an increased level of follistatin-3 identifies the subject as being suitable for treatment.

[00398] In certain embodiments, an increased level of follistatin-3 and an increased level of galectin-3 identifies the subject as being suitable for treatment. Methods for administering agents to a subject are known in the art.

[00399] In certain embodiments, a suitable practitioner may adjust the concentration of an agent administered to a subject based on the level of FSTL-3 determined and the particular condition.

[00400] Certain embodiments of the present disclosure provide a kit for performing a method as described herein.

[00401] In certain embodiments, the kit comprises one or more reagents for detecting follistatin-3.

[00402] In certain embodiments, the kit comprises one or more reagents for detecting galectin-3.

[00403] In certain embodiments, the kit comprises one or more reagents for detecting follistatin-3 and one or more reagents for detecting galectin-3.

[00404] In certain embodiments, the kit is an ELISA kit.

[00405] An ELISA kit for use in a method as described herein may, for example, contain one or more of the following: an agent for detection of FSTL-3 such as an anti- FSTL-3 antibody; an agent for detection of galectin-3, such as an anti- galectin-3 antibody; antibody-coated plates, standards, a primary detection antibody (typically biotinylated), a secondary detection reagent (eg streptavidin-URP), diluent buffers, wash buffers, substrate and stop solutions, controls; other reagents and instructions/methodology. Other components are contemplated.

[00406] Certain embodiments of the present disclosure provide a kit when used to identify or screen for a condition associated with cardiac fibrotic remodelling occurring in a subject, the kit comprising one or more reagents for detecting folli statin-like 3.

[00407] Reagents for detecting follistatin-like 3 are described herein. In certain embodiments, the reagents comprise one or more primary or secondary antibodies.

[00408] For example, a kit for immunological detection may, for example, include one or more of the following components: an agent for detection of FSTL-3 such as an anti- FSTL-3 antibody; antibody-coated plates, standards, a primary detection antibody (typically biotinylated), a secondary detection reagent (eg streptavidin-URP), diluent buffers, wash buffers, substrate and stop solutions, controls; other reagents and instructions/methodology.

[00409] In certain embodiments, the kit may further comprise one or more reagents for detecting galectin-3. Reagents for detecting galectin 3 are described herein.

[00410] Certain embodiments of the present disclosure provide a combination product for use in a method as described herein.

[00411] In certain embodiments, the combination product comprises one or more reagents for detecting folli statin-3.

[00412] In certain embodiments, the combination product comprises one or more reagents for detecting folli statin-3.

[00413] In certain embodiments, the combination product comprises one or more reagents for detecting follistatin-3 and one or more reagents for detecting galectin-3.

[00414] A combination product for immunological detection may, for example, include one or more of the following components: an agent for detection of FSTL-3 such as an anti-FSTL-3 antibody; an agent for detection of galectin-3, such as an anti- galectin-3 antibody; antibody-coated plates, standards, a primary detection antibody (typically biotinylated), a secondary detection reagent (eg streptavidin-HRP), diluent buffers, wash buffers, substrate and stop solutions, controls; other reagents and instructions/methodology.

[00415] Certain embodiments of the present disclosure provide a combination product when used to identify or screen for a condition associated with cardiac fibrotic remodelling occurring in a subject.

[00416] Certain exemplary embodiments are illustrated by some of the following examples. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.

EXAMPLE 1 - Follistatin-like 3 is associated with increased left ventricular mass in an ageing population

[00417] Background: The physiologic changes of human cardiac ageing include left ventricular hypertrophy (LVH) and cardiac fibrosis that can subsequently lead to the development of systolic, diastolic heart failure, and atrial fibrillation. To date, it remains completely unknown which biochemical factor(s) modulate ageing-associated changes in cardiovascular physiology.

[00418] In a normal ageing population, we sought to examine whether circulating FSTL-3 level is a predictor of increased left ventricular mass.

[00419] Plasma levels of FSLT-3 were measured by a commercially available ELISA kit for FSLT-3 in plasma. (R&D Systems, catalogue number DFLRG0).

[00420] Methods and Results: In 67 patients, age (68±6 yrs), without existing cardiovascular disease or previous antihypertensive therapy were studied. Left ventricular (LV) volumes and mass indexed to height^{2 7} (LVMI) were calculated from transthoracic echocardiography. Despite the absence of clinically-defined LVH, there was a significant relationship between high FSTL-3 levels and LV mass (R=0.5, pO.001, Figure 1). [00421] High FSTL-3 levels were also associated with age (R=0.3, p<0.05), BMI (R=0.4, p<0.01), and increased C-reactive protein (CRP) (R=0.3, p<0.05), while systolic BP (SBP) was not a significant predictor of increased FSTL-3 (R=0.2, p=0.1). On backward multiple linear regression indexed for age, gender, BMI, renal function, CRP, and SBP; increased FSTL-3 levels remained a predictor of increased LV mass (β=0.3, p=0.007).

[00422] Conclusions: In this ageing normotensive population free of established cardiovascular disease, FSTL-3 appeared to play a role in modulating early ageing- associated LVH development prior to any significant functional changes.

EXAMPLE 2 - Follistatin-like 3 is elevated in acute heart failure patients

[00423] Background: In this study, we sought to examine whether: 1) FSTL-3 is elevated in patients with heart failure (HF) vs. age-matched controls, and 2) FSTL-3 is elevated in patients with acute decompensated HF vs. those with chronic HF; and 3) FSTL-3 levels change 5 weeks after an acute HF episode.

[00424] Methods and Results:

[00425] We measured plasma levels of FSLT-3 in: 1) healthy ageing volunteers (n=67, age 68±6 yrs), 2) patients with acute HF (n=45, age 71±13), and 3) patients with chronic HF (n=28, age 71±10). Plasma FSTL-3 levels were significantly higher patients with acute HF (mean 16,220 ± 8645 pg/mL) vs, chronic HF (mean 10,205 ± 5636 pg/mL, p<0.01) patients vs. healthy ageing volunteers (mean 5601 ± 955.4 pg/ml, p<0.001) (one-way ANOVA with Bonferroni post hoc analysis) (Figure 2A). In 27 patients who were admitted to hospital due to acute decompensated HF, blood samples were taken to assess for FSTL3 levels at baseline and 5 weeks after treatment. While there was no difference in NT-proBNP levels in patients with acute HF upon admission vs. 5 weeks of follow-up (medians of 3997 pg/mL vs 2678 pg/mL respectively; p=0.4, Wilcoxon matched pairs rank test); FSTL-3 levels were significantly reduced (mean ±SD, 14,117 ± 7374 pg/mL vs. 12,744 ± 5573 pg/mL, p<0.05, paired t-test) (Figures 2B, 3A and 3B).

[00426] The levels of FSTL-3 measured were as follows:

[00427] Conclusions:

[00428] Plasma levels of FSTL-3 are increased in HF patients with a further incremental increase in those with acute versus chronic HF. These results suggest that FSTL-3 is a potential sensitive biomarker of changes in HF status. Furthermore, FSTL3 appears to be an even earlier and more consistent biomarker of response to anti-failure therapy compared to NT-proB P.

[00429] In 27 patients, treatment for HF was undertaken. The treatment was conventional anti-heart failure treatment (combination of beta-blockers, diuretics and renin-angiotensin-aldosterone system inhibitors) at the discretion of the treating cardiologist and in accordance with current guidelines and best practice.

[00430] In these patients with acute HF, follow-up FSTL3 and NT-proBNP were obtained after 5 weeks of treatment.

[00431] The results are shown in Figure 3.

[00432] FSTL3 levels were reduced in acute HF patients after 5 weeks of treatment, before changes in NT-proBNP. EXAMPLE 3 - Follistatin-like 3 predicts aortic root enlargement in patients with bicuspid aortic valve

[00433] Background: Bicuspid aortic valve (BAV) is the most common congenital heart defect, affecting up to 2% of the population. One of the major consequences of BAV is the development of progressive dilatation of the aortic root and ascending aorta, through aortic remodeling, that could lead to rupture. The pathophysiology of BAV- related aortopathy is heterogenous, and factors that predict ascending aorta dilatation in BAV remain largely unknown.

[00434] We sought to evaluate whether: 1) FSTL-3 is elevated in BAV, and 2) predicts aortic root dilatation in BAV.

[00435] Methods and Results:

[00436] The results are shown in Figure 4.

[00437] We compared plasma levels of FSTL-3 in patients with bicuspid aortic valve (n=41) and age-matched healthy volunteers (n=l l). FSTL-3 levels were significantly higher in those with BAV vs controls (median 5844 pg/mL vs. 4409 pg/mL, p=0.01). Aortic valve morphology, hemodynamic measurements, and ascending aortic diameter, were measured using transthoracic echocardiography. Univariate analyses in BAV patients showed significant relationships between high FSTL-3 levels: 1) increased ascending aortic size (R=0.4, p=0.01), sinus and tubular diameter (R=0.4, p=0.01), and aortic sinus diameter (R=0.3, p=0.04). FSLT-3 levels did not correlate with any of the hemodynamic measurements. On multivariate analysis, adjusted for age, gender, BMI, renal function, ACE inhibitor, and statin medications; male gender (β=0.6, p<0.01), lack of statin use (β=0.5, p=0.01), and high FSTL-3 (β=0.4, p=0.02) are significant correlates of increased ascending aortic size.

[00438] The concentrations of FSTL-3 measured were as follows:

[00439] Conclusions: Patients with BAV have increased FSTL-3 levels. FSTL-3 levels appear to be exclusively associated with aortopathy in BAV rather than a predictor of aortic valve hemodynamic changes. Strategies that aim to slow aortopathy in BAV should take into account FSTL-3 levels as part of the management of BAV-associated complications.

EXAMPLE 4 - FSTL-3 is a predictor of CRT response

[00440] Inclusion criteria: Patients in whom CRT +/- implantable cardioverter defibrillator insertion was planned were eligible for the study irrespective of the aetiology of underlying heart failure.

[00441] Exclusion criteria: (i) inability to attend for follow-up evaluation; (ii) treatment with inhibitors of platelet ADP receptor activation e.g. clopidogrel; (iii) anticipated limitation of exercise capacity due to non cardiac disease e.g. severe COPD; (iv) severe renal failure (creatinine>3.0mg/dl [265.2 μιηοΙ/L]) or on dialysis; (v) life expectancy< 1 year due to non cardiac causes; (vi) inability to give informed consent; (vii) permanent atrial fibrillation

[00442] The clinical characteristics of the patients were as follows:

[00443] We tested whether baseline FSTL-3 predicts improvement in cardiac function post CRT. The data is shown in Figure 5.

[00444] High FSTL3 is an independent correlate of lower change in 6 minute walk test (Figure 5A), and V0₂ max (maximum oxygen consumption) (Figure 5B), independent of age, gender, BMI, NYHA class, and renal function

EXAMPLE 5 - Elevated FSTL-3 is a predictor for new onset atrial fibrillation

[00445] The study involved 134 patients with AF admitted to cardiology (mean age 69±12 yrs). Of these patients, 34 patients diagnosed with new onset AF by ECG. [00446] We compared the plasma levels of FSTL-3 in the patients. The results are s hown in Figure 6. The concentrations of FSTL-3 measured were as follows:

[00447] As can be seen, increased FSTL-3 was associated with new onset atrial fibrillation.

EXAMPLE 6 - Galectin 3 predicts improvement in LV volumes post CRT [00448] Introduction

[00449] Cardiac Resynchronisation Therapy (CRT) is commonly used in the management of patients with chronic systolic heart failure (CHF). However, up to 30- 50% patients with severe CHF who receive CRT derive no benefit. The exact mechanisms underlying this phenomenon are poorly understood and there are no well- established biomarkers to predict the response to CRT.

[00450] We sought to determine the association between baseline galectin 3 levels and response to CRT in severe CHF has not been established.

[00451] Purpose [00452] To investigate the relationship between: i) galectin-3 levels and presence of CHF vs age-matched healthy population; and ii) baseline plasma galectin-3 levels and reverse remodeling in patients undergoing CRT implantation.

[00453] Methods and Results:

[00454] Plasma Gal-3 levels were assayed using a commercially available sandwich ELISA (R&D Systems, Catalog No. DGAL30).

[00455] Plasma Gal-3 levels were compared in 67 patients, age (68±6 yrs), without existing cardiovascular disease or previous antihypertensive therapy and 28 patients (aged 71.2±9.4) with predominantly severe CHF (70% NYHA class III or IV) and planned CRT irrespective of the aetiology of heart failure. Transthoracic echocardiogram (TTE) and blood collection for routine biochemistry, NT pro-BNP and galectin-3 levels were performed in the healthy cohort and in the severe CHF cohort prior to and 6 months post-CRT implantation. There was a significant increase in Gal-3 levels in patients with CHF vs healthy ageing (Figure 7A). However, there was no relationship between baseline Gal-3 levels and LV volumes (left ventricular end systolic volumes (LVESV) or end diastolic volumes (LVEDV) in either the healthy ageing cohort or those with CHF. Baseline Gal-3 levels correlated with significant improvement in LVESV (p=0.01) and LVEDV (p=0.02) in CHF patients post CRT (Figure 7B and 7C). On multivariate analyses, galectin 3 remained the only independent predictor of the improvements in LVESV (p=0.015) and LVEDV (p=0.02) post-CRT after adjustment for age, gender, NT pro-BNP and NYHA class.

[00456] The concentrations of galectin-3 measured were as follows:

[00457] Conclusion

[00458] Plasma galectin 3 is a promising biomarker of improvement of LV volumes in response to CRT. It has discriminatory potential between patients with severe symptomatic CHF and patients with normal physiological change in LV volumes.

EXAMPLE 6 - New onset atrial fibrillation is associated with elevated galectin-3 levels

[00459] Background: Atrial fibrillation (AF) is a common debilitating cardiac disorder with increased risk of stroke and cardiac mortality. Atrial remodeling and atrial fibrosis have been suggested to participate in the pathogenesis of AF. Galectin-3 is a secreted protein that has regulatory roles in fibrosis, inflammation and tissue repair. Several recent clinical studies have reported an association between circulating galectin 3 levels and cardiac remodeling as well as adverse clinical outcomes in patients with CHF. Galectin-3 levels have been shown to predict atrial remodeling and incidence of AF. Thus, in this study, we sought to examine whether differential Galectin-3 levels predict chronicity of AF in patients.

[00460] Methods and Results:

[00461] The results are shown in Figure 8.

[00462] 112 patients hospitalized with AF were evaluated (mean age 69±12 yrs). Of those, 34 patients (30.4%) were diagnosed with new onset AF. Plasma Galectin-3 levels were measured by commercially available ELISA assay. On univariate analyses, patients with new onset AF had significantly higher Galectin-3 levels vs those with chronic AF (mean 9.4ng/mL±3.3 vs. 8.0±3.5, p<0.05, respectively). High Galectin-3 levels were also significantly correlated with NT-proB P levels (R=0.3, p<0.01), CRP levels (R=0.3, p<0.01), and CHA₂DS₂VASc scores (R=0.2, p<0.05). On multivariate analysis, adjusting for age, gender, and creatinine, high galectin-3 levels remains an independent correlate of presence of new onset AF (β=0.2, p<0.05, CI (1.01, 1.53).

[00463] The concentrations of galectin-3 were as follows:

[00464] Conclusions: Recent onset of AF is associated with elevated galectin-3 levels. This suggests that Galectin-3 may be a new blood biomarker in AF with the potential to predict atrial fibrosis and remodeling. The extent of elevation of Galectin-3 levels in new onset AF correlates with parameters of stretch, inflammation and elevation of CHADSs score. Early elevation of Galectin-3 in new onset AF may be the precipitant for the inflammatory activation leading to development of long-term myocardial fibrosis in chronic AF.

EXAMPLE 7 - Galectin-3 levels is elevated in CHF vs age-matched healthy patients, and is more elevated in patients with acute HF

[00465] We measured plasma levels of GAL-3-3 in: 1) healthy ageing volunteers (n=67, age 68±6 yrs), 2) patients with acute HF (n=24, age 70±12), and 3) patients with chronic HF (n=28, age 71±9).

[00466] The results are shown in Fig [00467] We observed that plasma levels of GAL-3 are increased in HF patients. Acute IFF patients appear to have incremental increase in GAL-3 levels compared to those at chronic state. These results suggest that GAL-3 is a potential sensitive biomarker to detect changes in FIF.

EXAMPLE 8 - Elevated GAL-3 is a predictor for new onset atrial fibrillation

[00468] The study involved 132 patients with AF admitted to cardiology. Of these patients, 34 patients were diagnosed with new onset AF by ECG.

[00469] We compared the plasma levels of GAL-3 in the patients. The results are shown in Figure 10.

[00470] As can be seen, increased GAL-3 was associated with new onset atrial fibrillation.

EXAMPLE 9 - Combination of high FSTL3 and high Galectin-3 appears to be associated with lower change in 6MWT and VO? max

[00471] The results are shown in Figure 11.

[00472] A combination of high FSTL3 and high Galectin-3 appears to be associated with lower change in 6MWT and V0₂ max.

[00473] Patients with high FSTL3 and high Gal-3 appear to have lower reduction in LVEDV and LVESV with CRT (Figure 1 1).

[00474] A combination of high FSTL3 and high Galectin-3 appears to be associated with lower change in 6MWT and V0₂ max.

[00475] Patients with high FSTL3 and high Gal-3 appear to have lower reduction in LVEDV and LVESV with CRT (Figure 11). EXAMPLE 10 - High FSTL3 level is an independent correlate of increased risk of cardiovascular events as measured by the clinically used Framingham 10-year risk score

[00476] Framingham 10-year risk score is a clinical tool used to calculate a patient's risk of development cardiovascular events in 10 years time (Framingham Heart Study: https://www.framinghamheartstudy.org/risk-functions/cardiovascular-disease/10-year- risk.php)

[00477] In a cohort of n=253 subjects who volunteered, high FSTL-3 was found to be independently associated with high Framingham 10-year risk of: coronary heart disease, myocardial infarction, and cardiovascular diseases. Adjusted for body mass index, gender, renal function, and medications.

[00478] The data is as follows:

EXAMPLE 11 -Kits for determining level of human FSTL-3

[00479] An example of a kit for detecting FSTL-3 in plasma, serum and other biological fluids is as follows:

[00480] The kit may utilise a sandwich ELISA (enzyme-linked immunosorbent assay) kit, against human FSTL-3 utilising an antibody which recognizes human FSTL3.

[00481] The kit may use a 96-well enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of human FSTL3 in various samples, such plasma and serum.

[00482] Typically, such a kit may include the following components:

• Coated 96-well Strip Plate;

• Wash Buffer Concentrate;

• A standard (typically lyophilized);

• A biotinylated detection antibody;

• HRP-Streptavidin Conjugate;

• Substrate reagent(s)

• Stop solution; and

• Assay diluents

• Instructions

[00483] For analysis of plasma, typically plasma is collected using heparin, EDTA, or citrate as an anticoagulant. The samples are usually centrifuge samples for 15 minutes at 1000xg at 2-8°C within 30 minutes of collection. The supernatant is used for assaying.

[00484] For analysis of serum, a serum separator tube maybe used and samples are allowed to clot for 2 hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately lOOOxg. The supernatant is used for assaying.

[00485] For other types of biological samples, typically these samples may be processed by centrifuging samples for 20 minutes at lOOOxg to remove particulates, and collecting supernatant for assaying.

[00486] Determination of the level of FSTL-3 in a sample (see for example Han et al. (2014) Hypertens Pregnancy 33(3): 277-282) may then be used to inform a practitioner about the likelihood of a condition associated with cardiac fibrotic remodelling occurring in a subject, and also based on the level of FSTL-3 determined, to provide suitable treatment options and/or management strategies. Treatment strategies are described, for example, in "Textbook of Cardiology. Org" edited by Jonas de Jong; Fransje van der Walls (http://www.textbookofcardiology.org/wiki/Main_Page).

[00487] As used herein, the singular forms "a," "an," and "the" may refer to plural articles unless specifically stated otherwise.

[00488] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[00489] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.

[00490] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.

[00491] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.

[00492] Although the present disclosure has been described with reference to particular examples, it will be appreciated by those skilled in the art that the disclosure may be embodied in many other forms.

[00493] Future patent applications may be filed on the basis of the present application, for example by claiming priority from the present application, by claiming a divisional status and/or by claiming a continuation status. It is to be understood that the following claims are provided by way of example only, and are not intended to limit the scope of what may be claimed in any such future application. Nor should the claims be considered to limit the understanding of (or exclude other understandings of) the present disclosure. Features may be added to or omitted from the example claims at a later date.

Claims

1. A method of identifying a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling, the method comprising identifying an increased level of folli statin-like 3 in the subject.

2. The method according to claim 1, wherein the method comprises obtaining a biological sample from the subject and processing the sample to allow detection of the folli statin-like 3.

3. The method according to claim 2, wherein the biological sample is blood, plasma or serum.

4. The method according to any one of claims 1 to 3, wherein the condition is selected from one of cardiac hypertrophy, heart failure, response to cardiac resynchronisation therapy, paroxysmal atrial fibrillation, aortic root dilatation with aortic valve disease, chemotherapy-induced cardiovascular toxicity and stroke arising from atrial fibrillation.

5. The method according to any one of claims 1 to 4, wherein the condition is cardiac hypertrophy.

6. The method according to claim 5, wherein a plasma level of folli statin-like 3 greater than 6500 pg/ml is indicative that the subject is suffering from, or susceptible to, cardiac hypertrophy.

7. The method according to any one of claims 1 to 4, wherein the condition is heart failure.

8. The method according to claim 7, wherein the condition is heart failure and a plasma level of folli statin-like 3 greater than 7000 pg/ml is indicative that the subject is suffering from, or susceptible to, heart failure.

9. The method according to claim 7, wherein the condition is acute heart failure.

10. The method according to claim 9, wherein the condition is acute heart failure and a plasma level of follistatin-like 3 greater than 10000 pg/ml is indicative that the subject is suffering from, or susceptible to, acute heart failure.

11. The method according to claim 7, wherein the condition is chronic heart failure.

12. The method according to claim 11, wherein the condition is chronic heart failure and a plasma level of follistatin-like 3 greater than 7000 pg/ml is indicative that the subject is suffering from, or susceptible to, chronic heart failure.

13. The method according to claim 4, wherein the condition is response to cardiac resynchronisation therapy.

14. The method according to claim 13, wherein the condition is response to cardiac resynchronisation therapy and a plasma level of follistatin-like 3 of greater than 13000 pg/ml is indicative that the subject has a decreased likelihood of being responsive to cardiac resynchronisation therapy.

15. The method according to claim 13, wherein a plasma level of follistatin-like 3 of less than 1000 pg/ml is indicative that the subject has an increased likelihood of being responsive to cardiac resynchronisation therapy.

16. The method according to claim 4, wherein the condition is paroxysmal atrial fibrillation and/or stroke arising from atrial fibrillation.

17. The method according to claim 16, wherein the condition is paroxysmal atrial fibrillation and/or stroke arising from atrial fibrillation and a plasma level of follistatin- like 3 greater than 6600 pg/ml is indicative that the subject is suffering from, or susceptible to, paroxysmal atrial fibrillation and/or susceptible to a stroke arising from atrial fibrillation.

18. The method according to claim 4, wherein the condition is aortic root dilatation

19. The method according to claim 18, wherein the condition is aortic root dilatation and a plasma level of follistatin-like 3 greater than 9500 pg/ml is indicative that the subject is suffering from, or susceptible to, aortic root dilatation.

20. The method according to claim 4, wherein the condition is chemotherapy- induced cardiovascular toxicity.

21. The method according to claim 20, wherein the condition is chemotherapy- induced cardiovascular toxicity and a plasma level of follistatin-like 3 greater than 6500 pg/ml is indicative that the subject is suffering from, or susceptible to, chemotherapy- induced cardiovascular toxicity.

22. The method according to any one of claims 1 to 21, wherein the method further comprises identifying an increased level of galectin-3 in the subject.

23. The method according to any one of claims 13 to 15, wherein the method comprises identifying an increased plasma level of galectin-3 in the subject.

24. The method according to claim 23, wherein a plasma level of galectin-3 of greater than 15 ng/ml is indicative that the subject has a decreased likelihood of being responsive to cardiac resynchronisation therapy, and a plasma level of galectin-3 of less than 12.5 ng/ml is indicative that the subject has an increased likelihood of being responsive to cardiac resynchronisation therapy.

25. The method according to any one of claims 1 to 24, wherein the method further comprises determining the level of one or more of cholesterol, troponin, and B-type natriuretic peptide (BNP) and N-terminal pro B-type natriuretic peptide.

26. The method according to any one of claims 1 to 25, wherein the method comprises identifying the follistatin-like 3 by immunological detection.

27. The method according to any one of claims 1 to 26, wherein the method comprises using a computer processor means to process data associated with the level of the follistatin-like 3 to generate a likelihood and/or risk of the presence or absence of the condition in the subject.

28. A method of identifying a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling, the method comprising:

detecting follistatin-like 3 from the subject; and

identifying the subject as suffering from, or being susceptible to, a cardiac condition associated with cardiac fibrotic on the basis of the level of the follistatin-like 3 detected.

29. A method of identifying a subject suffering from, or susceptible to, a condition associated with cardiac fibrotic remodelling, the method comprising:

detecting follistatin-like 3 from the subject;

30. A method of screening for a condition associated with cardiac fibrotic remodelling occurring in a subject, the method comprising determining the level of follistatin-like 3 in the subject.

31. The method according to claim 30, wherein an increased level of follistatin-like 3 is indicative that the subject is suffering from, or being susceptible to, a condition associated with cardiac fibrotic remodelling.

32. A method of determining the likelihood or risk of a condition associated with cardiac fibrotic remodelling occurring in a subject, the method comprising:

determining the level of follistatin-like 3 from the subject; and

determining the likelihood or risk of the subject suffering from, or being susceptible to, a cardiac condition associated with cardiac fibrotic remodelling on the basis of the level of follistatin-3 determined.

33. The method according to claim 32, wherein an increased level of follistatin-like 3 is indicative of an increased likelihood or risk of the subject suffering from, or being susceptible to, a cardiac condition associated with cardiac fibrotic remodelling

34. The method according to any one of claims 30 to 33 for the diagnosis or prognosis of a condition associated with cardiac fibrotic remodelling in a subject.

35. A method of monitoring progression of a condition associated with cardiac fibrotic remodelling occurring in a subject, the method comprising determining the level of follistatin-like 3 in the subject and determining the extent of disease progression on the basis of the level of follistatin-like 3 determined.

36. A method of determining the responsiveness of a subject with chronic heart failure to cardiac resynchronisation therapy, the method comprising determining the level of follistatin-like 3 in the subject and determining the responsiveness of the subject to cardiac resynchronisation therapy.

37. A method of determining the likelihood or risk of a condition associated with cardiac fibrotic remodelling occurring in a subject, the method comprising:

determining the level of follistatin-like 3 from the subject; and

38. A kit for performing the method of any one of claims 1 to 37.

39. A kit when used to identify or screen for a condition associated with cardiac fibrotic remodelling occurring in a subject, the kit comprising one or more reagents for detecting follistatin-like 3.

40. The kit according to claim 38 or 39, wherein the kit further comprises one or more reagents for detecting galectin-3.

41. A method of preventing and/or treating a condition associated with cardiac fibrotic remodelling in a subject, the method comprising:

determining the level of follistatin-like 3 in the subject; and

42. A method of identifying a subject suitable for treatment for a condition associated with cardiac fibrotic remodelling in a subject, the method comprising:

determining the level of follistatin-like 3 in the subject; and

identifying the subject as a subject suitable for treatment on the basis of one or more clinical features of the subject and the level of follistatin-like 3 so determined.