MYL7 or a fragment thereof for the assessment of atrial heart muscle tissue damage

Abstract

The present invention relates to methods for assessing heart muscle tissue damage. The methods are based on the determination of the biomarker MyL7 in a sample from a subject. Moreover, the present invention relates to the use of the biomarker MyL7 (Myosin regulatory light chain 2, atrial isoform) or of at least one detection agent which specifically binds to said biomarker in at least one sample from a subject for assessing heart muscle tissue damage. The present invention further relates to a kit for assessing heart muscle tissue damage.

Description

[0001] MyL7 or a fragment thereof for the assessment of atrial heart muscle tissue damage

[0002] The present invention relates to methods for assessing heart muscle tissue damage. The methods are based on the determination of the biomarker MyL7 (Myosin regulatory light chain 2, atrial isoform) in a sample from a subject. Moreover, the present invention relates to the use of the biomarker MyL7 or of at least one detection agent which specifically binds to said biomarker in at least one sample from a subject for assessing heart muscle tissue damage. The present invention further relates to a kit for assessing heart muscle tissue damage.

[0003] Background section

[0004] Heart diseases are the leading causes of morbidity and mortality in developed countries. Currently, many biomarkers are used for the diagnosis of heart disease. For example, cardiac troponins and cardiac myosin binding protein C (cMyBPC) detect cardiac muscle damage. However, both biomarkers are not capable of differentiating between atrial heart muscle damage and ventricular heart muscle damage.

[0005] Ablation is a treatment in patients with atrial fibrillation that aims to correct abnormal heart rhythms by blocking electrical pathways in the heart. An ablation can be performed during cardiac surgery. Ablation lines are set in the atrium of the heart to prevent the transmission of electrical impulses that cause the atria to fibrillate. This ablation can be performed as endocardial ablation or as epicardial ablation. Both types of ablation damage heart muscle tissue.

[0006] Biomarkers for atrial heart muscle tissue damage are needed as they could be used in order to monitor whether an ablation therapy was successful. It is, thus, an important unmet need to assess the severity of atrial heart muscle tissue damage in order to plan therapies accordingly. In addition, as cardiac Troponins and cardiac myosin binding protein C are not capable of differentiating between atrial heart muscle damage and ventricular heart muscle damage, it is not possible to determine whether an increase of the biomarkers is caused i) by the ablation or i) by an additional heart muscle damage of the ventricle. Currently, patients are often subjected to further diagnostic measures in order, for example, to exclude a perioperative heart attack.

[0007] EP 3 775 914 Al discloses a method of diagnosing heart muscle tissue damage, preferably atrial heart muscle tissue damage, in a patient based on the biomarker MyBPHL. Published data in patients with endocardial ablation showed that MyBPHL values at admission to the ICU were observed to be about 3 to 4 times higher compared to the MyBPHL value prior to surgery (see Lahm, H., DreBen, M., Beck, N. et al. Myosin binding protein H-like (MYBPHL): a promising biomarker to predict atrial damage. Sci Rep 9, 9986 (2019)).

[0008] Doll analyzes the healthy human heart proteome by measuring 16 anatomical regions and three major cardiac cell types by high-resolution mass spectrometry-based proteomics (Doll, S., DreBen, M., Geyer, P.E. et al. Region and cell-type resolved quantitative proteomic map of the human heart. Nat Commun 8, 1469 (2017)).

[0009] MyL7 (myosin light chain 7, also known as Atrial Light Chain-2 (ALC-2) or Myosin regulatory light chain 2, atrial isoform (MLC2a)) is a protein that in humans is encoded by the MYL7 gene. It is expressed in the cardiac muscle atria in healthy individuals, where it functions to modulate cardiac development and contractility. MyL7 is known to be expressed in both atria (see Hailstones et al., Journal of Biological Chemistry, Volume 267, Issue 32, 1992, Pages 23295-23300).

[0010] Advantageously, it was shown in the studies underlying the present invention that biomarker MyL7 is a reliable marker for heart muscle tissue damage, in particular for atrial heart muscle tissue damage. For example, it was shown that the biomarker is increased in patients with atrial fibrillation, a disease related to modest atrial muscle tissue damage (see Example 2 and Figure 1). Moreover, the biomarker was increased after cardiac ablation (see Example 3 and Figure 2).

[0011] In contrast to published data of MyBPHL, the inventors observed in the same setting much higher elevations of MyL7 after cardiac ablation. Surprisingly, MyL7 values at admission to the ICU were observed to be about 180000 times higher compared to the MyL7 value prior to surgery (versus about 3 to 4 times with MyBPHL). Thus, the biomarker reliably allows for the detection of cardiac muscle damage. Interestingly, it was also shown in the studies underlying the present invention that the biomarker MyL7 can be used for determining the extent of heart muscle tissue damage, in particular of atrial heart muscle tissue damage. Specifically, it was shown that after endocardial ablation, the biomarker is much higher compared to epicardial ablation. Since endocardial ablation is known to cause more heart muscle tissue damage than epicardial ablation, the biomarker MyL7 correlates with extent of heart muscle tissue damage (see e.g. EP 3 775 914 Al). Further, the biomarker correlates with cardiac Troponin T which is an established marker of cardiac muscle damage (see e.g. Fig. 2A). Thus, the higher the amount of the marker MyL7, the higher the extent of cardiac injury (and vice versa). Therefore, MyL7 can be advantageously used as a marker for assessing the extent of heart muscle tissue damage.

[0012] The Figures show

[0013] Fig. 1 Results for patients of the MAPPING study: A) MyL7 in patients with paroxysmal atrial fibrillation (parAF), persistent atrial fibrillation (persAF) and patient in sinus rhythm (SR), B) MyL7 in patients with atrial fibrillation (AFib) and patient in sinus rhythm (SR), C) ROC curve for the biomarker MyL7, D) MyBPC in patients with atrial fibrillation (AFib) and patient in sinus rhythm (SR), E) ROC curve for the biomarker MyBPC.

[0014] Fig. 2 Results for patients of the MAZE study: A) The figure shows a normalized (with respect to the pre-surgery value, e.g. MYL7_normalized=(MYL7 at 2h) / (MYL7 pre-surgery)) biomarker trend line for different ablation procedures over the time. The plot is based on 52 patients: 32 with endocardial (left and right), 6 endocardial (left), 13 epicardial (left and right), 1 epicardial (right). The axis is fixed for all plots. In patients with endocardial ablation, the biomarker values rise much higher compared to epicardial ablations; B) as Figure 2A, but with a flexible axis; C) Example of a patient with left and right epicardial ablation. Normalized (with respect to the pre OP value) biomarker values before (pre OP) and after (arrival ICU, 2h-24h) the ablation procedure. D) Fig: Example of a patient with left and right endocardial ablation. Normalized (with respect to the pre OP value) biomarker values before (pre OP) and after (arrival ICU, 2h-24h) the ablation procedure.

[0015] Fig.3 Results for patients from the RRTAK study. MYL7 levels correspond to the degree of mitral insufficiency. A total of 49 patients were enrolled. 9 patients showed no, 30 patients showed Grad I, 8 patients showed Grad II and 2 patients showed Grad III mitral insufficiency. MYL7 levels in blood correlated with the degree of mitral insufficiency, whereby low levels were found in patients with no mitral insufficiency intermediate levels in Grad I and high levels in Grad II mitral insufficiency. Patient number for Grad III was too low.

[0016] Fig.4 Results for patients from the Mapping study. MYL7 levels correspond to the persistent atrial fibrillation and paroxysmal atrial fibrillation and sinus arrhythmia. A total 46 patients were enrolled. 26 showed sinus arrhythmia and low levels of MYL7, 11 patients showed paroxysmal atrial fibrillation ad low or slightly increased levels of MYL7. 9 patients showed persistent atrial fibrillation and increased level of MYL7.

[0017] Brief summary of the present invention

[0018] The present invention relates to a method for assessing heart muscle tissue damage, said method comprising the determination of the amount of the biomarker MyL7 in at least one sample from a subject, and the assessment of heart muscle tissue damage based on the amount of said biomarker.

[0019] In a preferred embodiment of said method of the present invention, the method further comprises the comparison of the amount of the biomarker to a reference amount.

[0020] The present invention further relates to a method of assessing heart muscle tissue damage, said method comprising the steps of: a) providing at least one sample from a subject, b) determining, in the at least one sample provided in step a), the amount of the biomarker MyL7 and, optionally, the amount of at least one further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC, and c) providing information on the determined amount of the biomarker MyL7 and optionally on the determined amount of the at least one further biomarker to a physician, thereby aiding in the assessment of heart muscle tissue damage.

[0021] The present invention further relates to a method assessing heart muscle tissue damage, comprising: a) providing an assay for the biomarker MyL7 and, optionally, at least one further assay for a further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC, and b) providing instructions for using of assay results obtained or obtainable by said assay(s) in the assessment of heart muscle tissue damage.

[0022] The present invention further relates to a computer-implemented method for assessing heart muscle tissue damage, comprising a) receiving, at a processing unit, a value for the amount of MyL7, and, optionally, at least one further value for the amount of at least one further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC, wherein said amount of MyL7 and, optionally, the amount of the at least one further biomarker have been determined in at least one sample from a subject, b) comparing, by said processing unit, the value or values received in step (a) to a reference or to references, and c) assessing heart muscle tissue damage based in the comparison step b).

[0023] The present invention further relates to a method of monitoring a cardiac surgery, comprising a) determining the amount of the biomarker MyL7 in a first sample obtained from a subject prior to a cardiac surgery, b) determining the amount of the biomarker MyL7 in a second sample obtained from a subject within 12 hours after said cardiac surgery, c) comparing the amount in the second sample to the amount in the first sample.

[0024] The present invention further relates to a method for determining the amount of the biomarker MyL7, and optionally the amount of at least one further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC, said method comprising a) providing at least one sample from a subject, wherein said sample has been obtained from said subject within 12 hours after cardiac surgery, and b) determining, in the at least one sample provided in step a), the amount of the biomarker MyL7 and, optionally, the amount of at least one further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC.

[0025] The present invention further relates to the use of the biomarker MyL7 (Myosin regulatory light chain 2, atrial isoform) or of at least one detection agent which specifically binds to said biomarker in at least one sample from a subject for assessing heart muscle tissue damage. In a preferred embodiment of this use, the use further comprises the use of i) a cardiac Troponin or a detection agent which specifically binds a cardiac Troponin and / or of ii) cMyBPC or a detection agent which specifically binds cMyBPC.

[0026] The present invention further relates to the use of at least one detection agent which specifically binds to MyL7 for determining the amount of MyL7 in a sample that has been obtained from a subject within 12 hours after cardiac surgery. In a preferred embodiment of this use, the use further comprises the use of i) a cardiac Troponin or a detection agent which specifically binds a cardiac Troponin for determining the amount of a cardiac Troponin and / or of ii) cMyBPC or a detection agent which specifically binds cMyBPC for determining the amount of cMyBPC.

[0027] The present invention further relates to a kit for assessing heart muscle tissue damage, said kit comprising at least one detection agent which specifically binds to MyL7 and optionally at least one detection agent which specifically binds to a cardiac Troponin and / or at least one detection agent which specifically binds to cMyBPC.

[0028] In a preferred embodiment of the methods, the uses or the kit of the present invention, the assessment of heart muscle tissue damage is selected from a) the diagnosis of heart muscle tissue damage, b) the differentiation between atrial and ventricular heart muscle tissue damage, c) the determination of the extent of heart muscle tissue damage, and d) the diagnosis of atrial arrhythmia.

[0029] In a preferred embodiment of the methods, the uses, or the kit of the present invention, the subject is a human subject.

[0030] In a preferred embodiment of the methods, the uses, or the kit of the present invention, the subject is suspected to suffer from heart muscle tissue damage. Preferably, the heart muscle tissue damage is atrial heart muscle damage.

[0031] In a preferred embodiment of the present invention, the sample is a blood, serum or plasma sample or interstitial fluid.

[0032] In a preferred embodiment of the present invention, the sample has been obtained after cardiac surgery, in particular after cardiac surgery involving the atria of the heart. Preferably, the cardiac surgery is cardiac ablation (for example endocardial or epicardial ablation), left atrial appendage closure, or interatrial shunt closure. Preferably, the sample has been obtained within 12 hours, such as within 8 hours, e.g. within 4 hours after surgery.

[0033] In a preferred embodiment of the methods of the present invention, the method further comprises the step of subjecting the patient to the cardiac surgery.

[0034] In a preferred embodiment of the present invention, the detection agent is an antibody or antigen-binding fragment thereof.

[0035] In a preferred embodiment of the present invention, the cardiac Troponin is cardiac Troponin T or I. In particular, the cardiac Troponin is cardiac Troponin T.

[0036] Detailed description of the present invention / Definitions

[0037] The present invention relates to a method for assessing heart muscle tissue damage, said method comprising the determination of the amount of the biomarker MyL7 in at least one sample from a subject, and the assessment of heart muscle tissue damage based on the amount of said biomarker in the at least one sample.

[0038] In a preferred embodiment of the method for assessing heart muscle tissue damage, the method further comprises the step of comparing the amount of the biomarker to a reference amount for MyL7. Preferably, the assessment of heart muscle tissue damage is based on the results of the comparison. In an embodiment, the method of the present invention thus comprises the further step of assessing heart muscle tissue damage in the subject based on the results of the comparison step.

[0039] In an embodiment, the present invention, thus, relates to a method for assessing heart muscle tissue damage, said method comprising a) determining the amount of the biomarker MyL7 in at least one sample from a subject, b) comparing the thus determined amount to a suitable reference amount, and c) assessing heart muscle tissue damage in the subject based on the results of the comparison step.

[0040] In an embodiment, the method of the present invention may further comprise the determination of the amount of at least one further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC. The determined amount(s) may be compared to a reference amount (or to reference amounts). The methods as referred to herein include methods which essentially consist of the mentioned steps or methods which include further steps. Moreover, the methods of the present invention are, preferably, in vitro methods. Moreover, they may comprise steps in addition to those explicitly mentioned. For example, further steps may relate to the determination of further markers and / or to sample pre-treatments or evaluation of the results obtained by the method. The methods of the present invention may be also used for monitoring, confirmation, and sub-classification of the subject. The methods may be carried out manually or assisted by automation. Preferably, step (a), (b) and / or (c) may in total or in part be assisted by automation, e.g., by a suitable robotic and sensory equipment for the determination in step (a) or a computer-implemented calculation in step (b).

[0041] As will be understood by those skilled in the art, the assessment described herein is usually not intended to be correct for all (i.e. 100%) of the subjects to be assessed. The term, typically, requires that a statistically significant portion of subjects can be correctly assessed (e.g., a cohort in a cohort study). Further, the actual assessment may comprise further steps such as the confirmation of the assessment. Thus, the assessment in the context of the present invention will aid the physician to assess heart muscle tissue damage. Accordingly, the term “assessing” in the context of the present invention preferably encompasses aiding the physician to assess heart muscle tissue damage.

[0042] Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well-known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. More preferably, at least 60%, at least 70%, at least 80% or at least 90% of the subjects of a population can be properly assessed by the methods of the present invention.

[0043] The term “heart muscle tissue damage” as used herein is well-known in the art, see e.g. EP 3 775 914 Al. As used herein, the term preferably refers to the damage of muscle tissue of the art. The term “damage” preferably includes the injury and death (necrosis) of cells present in the heart muscle tissue.

[0044] The human heart comprises four chambers consisting of the right and left atrium, and the right and left ventricle. Thus, there are two atria and two ventricles in the human heart. Preferably, the term “heart muscle tissue damage” as used herein refers to atrial heart muscle tissue damage, i.e. to damage in the atria. The “subject” as referred to herein is, preferably, a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non- human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the subject is a human subject.

[0045] In accordance with the present invention, the subject to be tested is, preferably, a subject who is suspected to suffer from heart muscle tissue damage. In particular, the subject is a subject who is suspected to suffer from atrial heart muscle damage.

[0046] For example, the subject who is suspected to suffer from heart muscle tissue damage preferably is a subject selected from:

[0047] • a subject who underwent cardiac surgery,

[0048] • a subject who is suspected to suffer from cardiac arrhythmia or who suffers from cardiac arrhythmia, preferably, sinus arrhythmia or atrial fibrillation,

[0049] • a subject who suffers from cardiopulmonary congestion,

[0050] • a subject who suffers from pulmonary embolism,

[0051] • a subject who suffers from cardiomyopathy (such as atrial cardiomyopathy), and

[0052] • a subject who suffers from pulmonary arterial hypertension

[0053] • a subject exhibiting mitral insufficiency.

[0054] In a preferred embodiment, the subject is a subject who underwent cardiac surgery, preferably cardiac surgery involving the atria. The term “cardiac surgery” is described elsewhere herein.

[0055] In another embodiment, the subject is a subject who is suspected to suffer from cardiac arrhythmia (preferably atrial flutter or atrial fibrillation). In yet another embodiment, the subject is a subject who suffers from cardiac arrhythmia (preferably atrial flutter or atrial fibrillation). In yet another embodiment, the subject is a subject who suffers from pulmonary embolism. In yet another embodiment, the subject is a subject who suffers from cardiopulmonary congestion. In yet another embodiment, the subject is a subject who suffers from cardiomyopathy (preferably atrial cardiomyopathy). In yet another embodiment, the subject is a subject who suffers from pulmonary arterial hypertension.

[0056] In a preferred embodiment of the invention, the term “assessing heart muscle tissue damage” relates to a) the diagnosis of heart muscle tissue damage, b) the differentiation between atrial and ventricular heart muscle tissue damage, c) the determination of the extent of heart muscle tissue damage, or d) the diagnosis of atrial arrhythmia.

[0057] Thus, in an embodiment of the present invention, the assessment is the diagnosis of heart muscle tissue damage. Thus, is it assessed whether the heart of the subject to be tested shows heart muscle tissue damage, or not. In other words, it is assessed whether the subjects suffers from heart muscle tissue damage, or not.

[0058] Accordingly, the present invention relates to a method for diagnosing heart muscle tissue damage, said method comprising a) determining the amount of the biomarker MyL7 in at least one sample from a subject, b) comparing the thus determined amount to a suitable reference amount, and c) diagnosing heart muscle tissue damage in the subject based on the results of the comparison step.

[0059] Preferably, an amount of the biomarker MyL7 in the sample from the subject which is above the reference amount indicates that the subject suffers from heart muscle tissue damage, whereas an amount of the biomarker MyL7 in the sample from the subject which is below the reference amount indicates that the subject does not suffer from heart muscle tissue damage. Preferably, the heart muscle tissue damage is atrial heart muscle tissue damage.

[0060] In another preferred embodiment, the present invention relates to a method for differentiating between atrial and ventricular heart muscle tissue damage, said method comprising a) determining the amount of the biomarker MyL7 in at least one sample from a subject, b) comparing the thus determined amount to a suitable reference amount, and optionally c) differentiating between atrial and ventricular heart muscle tissue damage based on the results of the comparison step.

[0061] The subject to be tested in accordance with the above method for differentiating between atrial and ventricular heart muscle tissue damage, preferably, suffers from heart muscle tissue damage. For example, the subject may have an elevated blood level of a cardiac Troponin and / or cMyBPC.

[0062] Preferably, an amount of the biomarker MyL7 in the sample from the subject which is above the reference amount indicates that the subject suffers from atrial heart muscle tissue damage, whereas an amount of the biomarker MyL7 in the sample from the subject which is below the reference amount indicates that the subject suffers from ventricular tissue damage.

[0063] In another preferred embodiment, the present invention relates to a method for determining the extent of heart muscle tissue damage, said method comprising a) determining the amount of the biomarker MyL7 in at least one sample from a subject, and b) determining the extent of heart muscle tissue damage in the subject based on the amount of the biomarker in the at least one sample.

[0064] In an embodiment, the extent of heart failure tissue damage (in particular atrial heart failure tissue damage) is determined in a subject who underwent cardiac surgery, in particular in a subject who underwent cardiac ablation to treat cardiac arrhythmia (in particular atrial fibrillation). Preferably, the subject is a human subject and the sample is a blood, serum or plasma sample. Preferred time points for obtaining the sample after the cardiac surgery are disclosed elsewhere herein. In step b), the extent of heart muscle tissue damage is, e.g., determined by comparing the amount of the marker(s) to a reference amount (or reference amounts). Based on the result of the comparison, the extent is determined. In an embodiment, the reference amount is a predetermined value, for example the amount of the marker in a sample obtained from the subject prior to the cardiac surgery. In some embodiments, the extent is determined by calculating a ratio of the amount of the marker after surgery to the amount of the marker prior to surgery.

[0065] In another preferred embodiment, the present invention relates to a method for diagnosing atrial fibrillation, said method comprising a) determining the amount of the biomarker MyL7 in at least one sample from a subject, b) comparing the thus determined amount to a suitable reference amount, and optionally c) diagnosing atrial fibrillation in the subject based on the results of the comparison step.

[0066] The subject to be tested in connection with the above method for diagnosing atrial fibrillation is preferably a subject who is suspected to suffer from atrial fibrillation (AF). Preferably, a subject who is suspected to suffer from AF, shall have shown at least one symptom of AF prior to carrying out the method for diagnosing AF, in particular before the sample has been obtained. Symptoms of atrial fibrillation include dizziness, fainting, shortness of breath and, in particular, heart palpitations. In some embodiments, the subject has experienced at least one symptom within 12 hours, such as within 8 hours before the sample to be tested has been obtained.

[0067] The atrial fibrillation to be diagnosed may be paroxysmal, permanent or persistent atrial fibrillation. Preferably, the atrial fibrillation to be diagnosed is permanent or persistent atrial fibrillation.

[0068] Preferably, an amount of the biomarker MyL7 in the sample from the subject which is above the reference amount indicates that the subject suffers from atrial fibrillation, whereas an amount of the biomarker MyL7 in the sample from the subject which is below the reference amount indicates that the subject does not suffer from atrial fibrillation.

[0069] The terms “paroxysmal”, “persistent” and “permanent” in connection with atrial fibrillation are well-known in the art. The American College of Cardiology (ACC), American Heart Association (AHA), and the European Society of Cardiology (ESC) propose the following classification system (see Fuster (2006) Circulation 114 (7): e257-354 which herewith is incorporated by reference in its entirety, see e.g. Figure 3 in the document): First detected AF, paroxysmal AF, persistent AF, and permanent AF. All people with AF are initially in the category called first detected AF. However, the subject may or may not have had previous undetected episodes. A subject suffers from permanent AF, if the AF has persisted for more than one year. In particular, conversion back to sinus rhythm does not occur (or only with medical intervention). A subject suffers from persistent AF, if the AF lasts more than 7 days. The subject may require either pharmacologic or electrical intervention to terminate Atrial Fibrillation. Thus persistent AF occurs in episodes, but the arrhythmia does typically not convert back to sinus rhythm spontaneously (i.e. without medical invention). Paroxysmal Atrial Fibrillation, preferably, refers to an intermittent episode of Atrial Fibrillation which lasts not longer than 7 days and terminates spontaneously (i.e. without medical intervention). In most cases of paroxysmal AF, the episodes last less than 24 hours. Thus, whereas paroxysmal atrial fibrillation terminates spontaneously, persistent atrial fibrillation does not end spontaneously and requires electrical or pharmacological cardioversion for termination, or other procedures, such as ablation procedures (Fuster (2006) Circulation 114 (7): e257-354). “Sinus arrhythmia” as referred to herein is a variation of the heart's sinus rhythm with an irregular rate. Preferably, the R-R interval is larger than 0.12 s.

[0070] Preferably, diagnosing atrial fibrillation as meant herein refers to differentiating between sinus arrhythmia, paroxysmal atrial fibrillation and persistent or permanent atrial fibrillation. More preferably, it refers to differentiating between sinus arrhythmia or paroxysmal atrial fibrillation on one hand and persistent or permanent atrial fibrillation on the other hand.

[0071] The term “sample” as used herein refers to any sample that under physiological conditions comprises the first, second and / or third biomarkers referred to herein. More typically, the sample is a body fluid sample, e.g. a blood sample or sample derived therefrom (e.g. serum or plasma), a urine sample, interstitial fluid, a saliva sample, a lymphatic fluid sample or the like. Most typically, said sample is a blood, serum or plasma sample.

[0072] Further, it is envisaged that a blood sample is a dried blood spot sample. Dried blood spot samples can be obtained by applying drops of blood onto absorbent filter paper. The blood is allowed to thoroughly saturate the paper and is air-dried for several hours. The blood may have been drawn by a lancet from the subject to be tested, e.g. from the finger.

[0073] In a preferred embodiment, the sample is a blood (i.e. whole blood), serum or plasma sample. Serum is the liquid fraction of whole blood that is obtained after the blood is allowed to clot. For obtaining the serum, the clot is removed by centrifugation and the supernatant is collected. Plasma is the acellular fluid portion of blood. For obtaining a plasma sample, whole blood is collected in anticoagulant-treated tubes (e.g. citrate-treated or EDTA-treated tubes). Cells are removed from the sample by centrifugation and the supernatant (i.e. the plasma sample) is obtained.

[0074] Blood samples include capillary blood samples. Such samples can be obtained, e.g., from a puncture on the finger.

[0075] In a preferred embodiment of the present invention, the sample has been obtained after cardiac surgery. This allows for assessing whether the cardiac surgery has caused heart muscle tissue damage, in partial atrial heart muscle tissue damage.

[0076] The cardiac surgery may be any kind of surgery. In particular the cardiac surgery is a cardiac surgery involving the atria of the heart.

[0077] In a preferred embodiment, the cardiac surgery is cardiac ablation. For example, the cardiac ablation is endocardial ablation. Alternatively, the cardiac ablation is epicardial ablation. Thus, the subject to be tested is a subject suffering from atrial fibrillation who has undergone a cardiac ablation. Alternatively, the subject to be tested is a subject suffering from atrial flutter or atrial tachycardia who has undergone cardiac ablation. Preferably, the cardiac ablation has been carried out to treat atrial fibrillation. In another preferred embodiment, the cardiac surgery is left atrial appendage closure. In yet another preferred embodiment, the cardiac surgery is interatrial shunt closure.

[0078] Preferably, the sample has been obtained within 12 hours after the surgery, more preferably within 8 hours and even more preferably within 4 hours after surgery, and most preferably, within 3 hours after surgery.

[0079] In another preferred embodiment, the sample has been obtained at least one hour, but no later than 8 hours after cardiac surgery. In yet another preferred embodiment, the sample has been obtained at least one hour, but no later than 4 hours after cardiac surgery. In yet another preferred embodiment, the sample has been obtained at least one hour, but no later than 3 hours after cardiac surgery.

[0080] Further, it is envisaged that the sample has been obtained at a predetermined time, i.e. at a fixed time, after cardiac surgery (but within 12 hours after surgery). For example, the sample can be obtained, about one hour, at least two hours, at least three hours, at least four hours, at least five hours, at least six hours, at least seven hours or at least eight hours after completion of surgery.

[0081] Moreover, it is envisaged to obtain more than one sample within 12 hours after surgery. For example, it is envisaged to obtain two, three, four or more samples. This can be done in order to determine the time course of the amount of the biomarkers. Preferably, the samples have been obtained at least 29 minutes apart, for example at least 59 minutes apart.

[0082] The end of the surgery typically is the completion of the surgery. In an embodiment, the completion is at the time point at which the subject is admitted to the ICU.

[0083] A sample may have been also obtained prior to surgery. As explained herein further below, this sample may serve as reference sample.

[0084] In accordance with the present invention, the amount of the biomarker MyL7 (myosin light chain 7) shall be determined. MyL7 is a protein that in humans is encoded by the MYL7 gene. The protein is also known as Atrial Light Chain-2 (ALC-2), as Myosin regulatory light chain 2, atrial isoform (MLC2a), or a MyL2A. The protein has a molecular weight of about 19.4 kDa and comprises 175 amino acids. It is an EF hand protein that binds to the neck region of alpha myosin heavy chain. The sequence of MyL7 is well known in the art. It can be e.g. assessed under UniProt accession number Q01449 (MLRA HUMAN). The biomarker is known to be expressed in both atria (see Hailstones et al., Journal of Biological Chemistry, Volume 267, Issue 32, 1992, Pages 23295-23300).

[0085] In various embodiments of the methods described herein, the method further comprises, in the at least one sample from the subject, determining the level of at least one further biomarker for heart muscle tissue damage. Preferably, the at least one further biomarker is a cardiac Troponin and / or cMyBPC. The further marker(s) may be compared to a reference amount (or to reference amounts).

[0086] Preferably, an increase of the biomarker(s) as compared to the reference amount(s) is indicative for a subject who suffers from heart muscle tissue damage (or atrial fibrillation). Preferably, a decrease of the biomarker(s) as compared to the reference amount(s) is indicative for a subject who does not suffer from heart muscle tissue damage (or atrial fibrillation).

[0087] The term “cardiac Troponin” refers to all Troponin isoforms expressed in cells of the heart and, preferably, the subendocardial cells. These isoforms are well characterized in the art as described, e.g., in Anderson 1995, Circulation Research, vol. 76, no. 4: 681-686 and Ferrieres 1998, Clinical Chemistry, 44: 487-493. Preferably, Troponin refers to Troponin T and / or Troponin I. Accordingly, both Troponins may be determined in the method of the present invention together, i.e. simultaneously or sequentially, or individually, i.e. without determining the other isoform at all. Amino acid sequences for human Troponin T and human Troponin I are disclosed in Anderson, loc cit and Ferrieres 1998, Clinical Chemistry, 44: 487-493. The term “cardiac Troponin” encompasses also variants of the aforementioned specific Troponins, i.e., preferably, of Troponin T or Troponin I.

[0088] In a preferred embodiment, the term “cardiac Troponin” refers to cardiac Troponin T (which was measured in the Examples). In another preferred embodiment, the term “cardiac Troponin” refers to cardiac Troponin I.

[0089] Myosin-binding protein C is a myosin-associated protein found in the cross-bridge-bearing zone (C region) of A bands in striated muscle. In accordance with the present invention, the amount of the cardiac isoform, i.e. cMyBPC (cardiac Myosin binding protein C) shall be determined (also referred to as MYBPC3, CMD1MM, CMH4, FHC, LVNC10, MYBP-C, Myosin binding protein C, cardiac, cMyBP-C, myosin binding protein C3). cMyBPC is produced in heart muscle. It is encoded by the MYBPC3 gene. Further information on human cMYBPC can be found in the UniProtKB database under accession number Q14896 (MYPC3 HUMAN). The term “determining” as used herein refers to qualitative and quantitative determination of the biomarkers referred to in accordance with the present invention, i.e. the term encompasses the determination of the presence or absence or the determination of the absolute or relative amount of said biomarkers. Preferably, the term “determining” the amount of a biomarker as referred to herein refers to the quantification of the biomarker, e.g. to measuring the level of the biomarker in the sample, employing appropriate methods of detection described elsewhere herein. The terms “measuring” and “determining” are used herein interchangeably.

[0090] Typically, the amount of a biomarker as referred to in accordance with the present invention can be determined by immunoassays using sandwich, competition, or other assay formats. Said assays will develop a signal which is indicative for the presence or absence or the amount of a biomarker.

[0091] Measuring the amount of a biomarker may, preferably, comprise the steps of (a) contacting the polypeptide with an agent that specifically binds said polypeptide, (b) (optionally) removing non-bound agent, (c) measuring the amount of bound binding agent, i.e. the complex of the agent formed in step (a). According to a preferred embodiment, said steps of contacting, removing and measuring may be performed by an analyzer unit. According to some embodiments, said steps may be performed by a single analyzer unit of said system or by more than one analyzer unit in operable communication with each other. For example, according to a specific embodiment, said system disclosed herein may include a first analyzer unit for performing said steps of contacting and removing and a second analyzer unit, operably connected to said first analyzer unit by a transport unit (for example, a robotic arm), which performs said step of measuring.

[0092] The agent which specifically binds the biomarker (herein also referred to as “binding agent”) may be coupled covalently or non-covalently to a label allowing detection and measurement of the bound agent. Labeling may be done by direct or indirect methods. Direct labeling involves coupling of the label directly (covalently or non-covalently) to the binding agent. Indirect labeling involves binding (covalently or non-covalently) of a secondary binding agent to the first binding agent. The secondary binding agent should specifically bind to the first binding agent. Said secondary binding agent may be coupled with a suitable label and / or be the target (receptor) of a tertiary binding agent binding to the secondary binding agent. Suitable secondary and higher order binding agents may include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). The binding agent or substrate may also be "tagged" with one or more tags as known in the art. Such tags may then be targets for higher order binding agents. Suitable tags include biotin, digoxygenin, His-Tag, Glutathion-S-Transferase, FLAG, GFP, myc-tag, influenza A virus haemagglutinin (HA), maltose binding protein, and the like. In the case of a peptide or polypeptide, the tag is preferably at the N-terminus and / or C-terminus. Suitable labels are any labels detectable by an appropriate detection method. Typical labels include gold particles, latex beads, acridan ester, luminol, ruthenium complexes, iridium complexes, enzymatically active labels, radioactive labels, magnetic labels ("e.g. magnetic beads", including paramagnetic and superparamagnetic labels), and fluorescent labels. Enzymatically active labels include e.g. horseradish peroxidase, alkaline phosphatase, beta- Galactosidase, Luciferase, and derivatives thereof. Suitable substrates for detection include di-amino-benzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, avail-able as ready-made stock solution from Roche Diagnostics), CDP-Star™ (Amersham Bio-sciences), ECF™ (Amersham Biosciences). A suitable enzyme-substrate combination may result in a colored reaction product, fluorescence or chemoluminescence, which can be measured according to methods known in the art (e.g. using a light-sensitive film or a suit-able camera system). As for measuring the enzymatic reaction, the criteria given above apply analogously. Typical fluorescent labels include fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red, Fluorescein, and the Alexa dyes (e.g. Alexa 568). Further fluorescent labels are available e.g. from Molecular Probes (Oregon). Also the use of quantum dots as fluorescent labels is contemplated. A radioactive label can be detected by any method known and appropriate, e.g. a light-sensitive film or a phosphor imager.

[0093] The amount of a polypeptide may be, also preferably, measured as follows: (a) contacting a solid support comprising a binding agent for the polypeptide as described elsewhere herein with a sample comprising the peptide or polypeptide and (b) measuring the amount of peptide or poly-peptide which is bound to the support. Materials for manufacturing supports are well-known in the art and include, inter alia, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and / or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction trays, plastic tubes etc.

[0094] In yet an aspect the sample is removed from the complex formed between the binding agent and the at least one marker prior to the measurement of the amount of formed complex. Accordingly, in an aspect, the binding agent may be immobilized on a solid support. In yet an aspect, the sample can be removed from the formed complex on the solid support by applying a washing solution. “Sandwich assays” are among the most useful and commonly used assays encompassing a number of variations of the sandwich assay technique. Briefly, in a typical assay, an unlabeled (capture) binding agent is immobilized or can be immobilized on a solid substrate, and the sample to be tested is brought into contact with the capture binding agent. After a suitable period of incubation, for a period of time sufficient to allow formation of a binding agent-biomarker complex, a second (detection) binding agent labeled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of binding agent-biomarker-labeled binding agent. Any unreacted material may be washed away, and the presence of the biomarker is determined by observation of a signal produced by the reporter molecule bound to the detection binding agent. The results may either be qualitative, by simple observation of a visible signal, or may be quantitated by comparison with a control sample containing known amounts of biomarker.

[0095] The incubation steps of a typical sandwich assays can be varied as required and appropriate. Such variations include for example simultaneous incubations, in which two or more of binding agent and biomarker are co-incubated. For example, both, the sample to be analyzed and a labeled binding agent are added simultaneously to an immobilized capture binding agent. It is also possible to first incubate the sample to be analyzed and a labeled binding agent and to thereafter add an antibody bound to a solid phase or capable of binding to a solid phase.

[0096] The formed complex between a specific binding agent and the biomarker shall be proportional to the amount of the biomarker present in the sample. It will be understood that the specificity and / or sensitivity of the binding agent to be applied defines the degree of proportion of at least one marker comprised in the sample which is capable of being specifically bound. Further details on how the measurement can be carried out are also found elsewhere herein. The amount of formed complex shall be transformed into an amount of the biomarker reflecting the amount indeed present in the sample.

[0097] The terms "binding agent", “specific binding agent”, “analyte-specific binding agent”, “detection agent” and “agent that specifically binds to a biomarker” are used interchangeably herein. Preferably it relates to an agent that comprises a binding moiety which specifically binds the corresponding biomarker. Examples of “binding agents” or “agents” are a nucleic acid probe, nucleic acid primer, DNA molecule, RNA molecule, aptamer, antibody, antibody fragment, peptide, peptide nucleic acid (PNA) or chemical compound. A preferred agent is an antibody, or antigen-binding fragment thereof, which specifically binds to the biomarker to be measured. The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity (i.e. antigen-binding fragments thereof). Preferably, the antibody is a polyclonal antibody. More preferably, the antibody is a monoclonal antibody.

[0098] The term “specific binding” or “specifically bind” refers to a binding reaction wherein binding pair molecules exhibit a binding to each other under conditions where they do not significantly bind to other molecules. The term “specific binding” or “specifically binds”, when referring to a protein or peptide as biomarker, refers to a binding reaction wherein a binding agent binds to the corresponding biomarker with an affinity of at least 10'7M. The term “specific binding” or “specifically binds” preferably refers to an affinity of at least I O'8M or even more preferred of at least 10'9M for its target molecule. The term “specific” or “specifically” is used to indicate that other molecules present in the sample do not significantly bind to the binding agent specific for the target molecule.

[0099] The determination of a biomarker as set forth herein may comprise mass spectrometry (MS) which is carried out after the separation step (e.g. by LC or HPLC). Mass spectrometry as used herein encompasses all techniques which allow for the determination of the molecular weight (i.e. the mass) or a mass variable corresponding to a compound, i.e. a biomarker, to be determined in accordance with the present invention. Preferably, mass spectrometry as used herein relates to GC-MS, LC-MS, direct infusion mass spectrometry, FT-ICR-MS, CE- MS, HPLC-MS, quadrupole mass spectrometry, any sequentially coupled mass spectrometry such as MS-MS or MS-MS-MS, ICP-MS, Py-MS, TOF or any combined approaches using the aforementioned techniques. How to apply these techniques is well known to the person skilled in the art. Moreover, suitable devices are commercially available. More preferably, mass spectrometry as used herein relates to LC-MS and / or HPLC-MS, i.e. to mass spectrometry being operatively linked to a prior liquid chromatography separation step. Preferably, the mass spectrometry is tandem mass spectrometry (also known as MS / MS). Tandem mass spectrometry, also known as MS / MS involves two or more mass spectrometry steps, with a fragmentation occurring in between the stages. The mass spectrometers are coupled to the chromatographic device. The sample that has been separated by a chromatography is sorted and weighed in the first mass spectrometer, then fragmented by an inert gas in the collision cell, and a piece or pieces sorted and weighed in the second mass spectrometer. The fragments are sorted and weighed in the second mass spectrometer. Identification by MS / MS is more accurate. In an embodiment, mass spectrometry as used herein encompasses quadrupole MS. Most preferably, said quadrupole MS is carried out as follows: a) selection of a mass / charge quotient (m / z) of an ion created by ionisation in a first analytical quadrupole of the mass spectrometer, b) fragmentation of the ion selected in step a) by applying an acceleration voltage in an additional subsequent quadrupole which is filled with a collision gas and acts as a collision chamber, c) selection of a mass / charge quotient of an ion created by the fragmentation process in step b) in an additional subsequent quadrupole, whereby steps a) to c) of the method are carried out at least once and analysis of the mass / charge quotient of all the ions present in the mixture of substances as a result of the ionisation process, whereby the quadrupole is filled with collision gas but no acceleration voltage is applied during the analysis. Details on said most preferred mass spectrometry to be used in accordance with the present invention can be found in W02003 / 073464.

[0100] More preferably, said mass spectrometry is liquid chromatography (LC) MS such as high performance liquid chromatography (HPLC) MS, in particular HPLC-MS / MS. Liquid chromatography as used herein refers to all techniques which allow for separation of compounds (i.e. metabolites) in liquid or supercritical phase.

[0101] For mass spectrometry, the analytes in the sample are ionized in order to generate charged molecules or molecule fragments. Afterwards, the mass-to-charge of the ionized analyte, in particular of the ionized biomarkers, or fragments thereof is measured. Prior to the ionization, the sample may be subjected to cleavage with a protease, e.g. with trypsin. The protease cleaves the protein biomarkers into smaller fragments.

[0102] Thus, the mass spectrometry step preferably comprises an ionization step in which the biomarkers to be determined are ionized. Of course, other compounds present in the sample / elulate are ionized as well. Ionization of the biomarkers can be carried out by any method deemed appropriate, in particular by electron impact ionization, fast atom bombardment, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), matrix assisted laser desorption ionization (MALDI).

[0103] In a preferred embodiment, the ionization step (for mass spectrometry) is carried out by electrospray ionization (ESI). Accordingly, the mass spectrometry is preferably ESLMS (or if tandem MS is carried out: ESLMS / MS). Electrospray is a soft ionization method which results in the formation of ions without breaking any chemical bonds.

[0104] The term “amount” as used herein refers to the absolute amount of a compound referred to herein, the relative amount or concentration of the said compound as well as any value or parameter which correlates thereto or can be derived therefrom. Such values or parameters comprise intensity signal values from all specific physical or chemical properties obtained from the said compounds by direct measurements, e.g., intensity values in mass spectra or NMR spectra. Moreover, encompassed are all values or parameters which are obtained by indirect measurements specified elsewhere in this description, e.g., response levels determined from biological read out systems in response to the compounds or intensity signals obtained from specifically bound ligands. It is to be understood that values correlating to the aforementioned amounts or parameters can also be obtained by all standard mathematical operations.

[0105] The term “comparing” as used herein refers to comparing the amount of the biomarker in the sample from the subject with the reference amount of the biomarker specified elsewhere in this description. It is to be understood that comparing as used herein usually refers to a comparison of corresponding parameters or values, e.g., an absolute amount is compared to an absolute reference amount while a concentration is compared to a reference concentration or an intensity signal obtained from the biomarker in a sample is compared to the same type of intensity signal obtained from a reference sample. The comparison may be carried out manually or computer-assisted. Thus, the comparison may be carried out by a computing device. The value of the measured or detected amount of the biomarker in the sample from the subject and the reference amount can be, e.g., compared to each other and the said comparison can be automatically carried out by a computer program executing an algorithm for the comparison. The computer program carrying out the said evaluation will provide the desired assessment in a suitable output format. For a computer-assisted comparison, the value of the measured amount may be compared to values corresponding to suitable references which are stored in a database by a computer program. The computer program may further evaluate the result of the comparison, i.e. automatically provide the desired assessment in a suitable output format. For a computer-assisted comparison, the value of the measured amount may be compared to values corresponding to suitable references which are stored in a database by a computer program. The computer program may further evaluate the result of the comparison, i.e. automatically provides the desired assessment in a suitable output format.

[0106] In a preferred embodiment of the present invention, the assessment of heart of muscle tissue damage is the diagnosis of atrial heart muscle tissue in a human subject, wherein the sample is a blood, serum of plasma sample that has been obtained after the subject to be tested underwent cardiac ablation, in particular within 12 hours after the subject to be tested underwent cardiac ablation. In another preferred embodiment of the present invention, atrial heart muscle tissue damage is diagnosed, wherein the sample is a blood, serum of plasma sample that has been obtained within 8 hours after the subject to be tested underwent cardiac ablation. In yet another preferred embodiment of the present invention, atrial heart muscle tissue damage is diagnosed, wherein the sample is a blood, serum of plasma sample that has been obtained within 4 hours after the subject to be tested underwent cardiac ablation. Preferably, the cardiac ablation has been carried out to treat atrial arrhythmia, in particular atrial fibrillation in said subject.

[0107] In a further preferred embodiment of the present invention, the assessment of heart muscle tissue damage is the determination of the extent of atrial heart muscle tissue in a human subject, wherein the sample is a blood, serum of plasma sample that has been obtained after the subject to be tested underwent cardiac ablation, in particular within 12 hours after the subject to be tested underwent cardiac ablation. In another preferred embodiment of the present invention, the extent of atrial heart muscle tissue damage is determined, wherein the sample is a blood, serum of plasma sample that has been obtained within 8 hours after the subject to be tested underwent cardiac ablation. In yet another preferred embodiment of the present invention, the extent of atrial heart muscle tissue damage is determined, wherein the sample is a blood, serum of plasma sample that has been obtained within 4 hours after the subject to be tested underwent cardiac ablation. Preferably, the cardiac ablation has been carried out to treat atrial arrhythmia, in particular atrial fibrillation.

[0108] In a further preferred embodiment of the present invention, the assessment of heart muscle tissue damage is the diagnosis of atrial fibrillation in a human subject suspected to suffer from atrial fibrillation, wherein the sample is a blood, serum of plasma sample.

[0109] The definitions and explanations provided herein above apply mutatis mutandis to the following methods, uses, kits and devices of the present invention.

[0110] The present invention further relates to a method of assessing heart muscle tissue damage, said method comprising the steps of: a) providing at least one sample from a subject, b) determining, in the at least one sample provided in step a), the amount of the biomarker MyL7 and, optionally, the amount of at least one further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC, and c) providing information on the determined amount of the biomarker MyL7 and optionally on the determined amount of the at least one further biomarker to a physician, thereby aiding in the assessment of heart muscle tissue damage. Step a) of the aforementioned method (providing at least one sample from a subject) does not encompass the drawing of the sample from the subject. Preferably, the sample is obtained by receiving a sample from said subject. Thus, the sample can have been delivered, e.g. to a laboratory which carries out the determination of the biomarker (s).

[0111] The physician shall be the attending physician, i.e. the physician who requested the determination of the biomarker(s). The aforementioned method shall aid the attending physician in the assessment of heart muscle tissue damage. Thus, the method does not encompass the assessment as referred to above in connection with the method of heart muscle tissue damage. The information on the amount of the biomarker(s) can be provided electronically.

[0112] The present invention further relates to a method assessing heart muscle tissue damage, comprising: a) providing an assay for the biomarker MyL7 and, optionally, at least one further assay for a further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC, and b) providing instructions for using of assay results obtained or obtainable by said assay(s) in the assessment of heart muscle tissue damage.

[0113] The instructions shall contain a protocol for carrying out the method of assessing heart muscle tissue damage as described herein above. Further, the instructions shall contain at least one value for a reference amount for the biomarker MyL7 and optionally at least one value for a reference amount for the further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC.

[0114] The “assay” is preferably a kit adapted for determining the amount of the biomarker. The term “kit” is explained herein below.

[0115] The assay result obtained or obtainable by said test, is the value for the amount of the biomarker(s).

[0116] The present invention further relates to a computer-implemented method for assessing heart muscle tissue damage, comprising a) receiving, at a processing unit, a value for the amount of MyL7, and, optionally at least one further value for the amount of at least one further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC, wherein said amount of MyL7 and, optionally, the amount of the at least one further biomarker have been determined in at least one sample from a subject, b) comparing, by said processing unit, the value or values received in step (a) to a reference or to references, and c) assessing heart muscle tissue damage based in the comparison step b).

[0117] The above-mentioned method is a computer-implemented method. Preferably, all steps of the computer-implemented method are performed by one or more processing units of a computer (or computer network). Thus, the assessment in step (c) is carried out by a processing unit. Preferably, said assessment is based on the results of step (b).

[0118] The value or values received in step (a) shall be derived from the determination of the amount of the biomarker from a subject as described elsewhere herein. Preferably, the value is a value for the concentration of the biomarker. The value will be typically received by the processing unit by uploading or sending the value to the processing unit. Alternatively, the value can be received by the processing unit by inputting the value via a user interface.

[0119] In an embodiment of the aforementioned method, the reference (or references) set forth in step (b) is (are) established from a memory. Preferably, a value for the reference is established from the memory.

[0120] In an embodiment of the aforementioned computer-implemented method of the present invention, the result of the assessment made in step c) is provided via a display, configured for presenting result.

[0121] In an embodiment of the aforementioned computer-implemented method of the present invention, the method may comprise the further step of transferring the information on the assessment made in step c) to the subject’s electronic medical records.

[0122] In an embodiment of the methods for assessing heart muscle tissue damage, the methods further comprise a step of recommending and / or initiating a therapy for atrial fibrillation based on the results of the assessment. For example, a therapy is recommended or initiated, if it is diagnosed that the subject suffers from AF. For example, treatment with at least one anticoagulant can be recommended or initiated. In a preferred embodiment, the at least one anticoagulant is selected from the group consisting of heparin, a coumarin derivative (i.e. a vitamin K antagonist), in particular warfarin or dicumarol, oral anticoagulants, in particular dabigatran, rivaroxaban or apixaban, tissue factor pathway inhibitor (TFPI), antithrombin III, factor IXa inhibitors, factor Xa inhibitors, inhibitors of factors Va and Villa and thrombin inhibitors (anti-IIa type). Alternatively, a cardiac ablation is recommended or initiated, if it is diagnosed that the subject suffers from AF.

[0123] In accordance with the present invention, the amount of the biomarker MyL7 (and optionally the at least one further biomarker) shall be compared to a reference, i.e. to a reference amount (or to reference amounts). Accordingly, the reference is preferably a reference amount. The terms “reference amount” or “reference” are well understood by the skilled person. It is to be understood that the reference amount shall allow for aiding in the assessment of heart muscle tissue damage, such as in the diagnosis of heart muscle tissue damage, the determination of the extent of heart failure tissue damage, and / or the diagnosis of atrial arrhythmia. For example, in connection with the method for diagnosing heart muscle tissue damage, the reference amount preferably refers to an amount which allows for allocation of a subject into either (i) the group of subjects suffering from heart muscle tissue damage, or (ii) the group of subjects not suffering from heart muscle tissue damage. For example, in connection with the method for diagnosing of atrial arrhythmia, the reference amount preferably refers to an amount which allows for allocation of a subject into either (i) the group of subjects suffering from atrial arrhythmia and / or (ii) the group of subjects not suffering from atrial arrhythmia.

[0124] Reference amounts can, in principle, be calculated for a cohort of subjects as specified above based on the average or mean values for a given biomarker by applying standard methods of statistics. In particular, accuracy of a test such as a method aiming to diagnose an event, or not, is best described by its receiver-operating characteristics (ROC) (see especially Zweig MH. et al., Clin. Chem. 1993;39:561-577). The ROC graph is a plot of all the sensitivity versus specificity pairs resulting from continuously varying the decision threshold over the entire range of data observed. The clinical performance of a diagnostic method depends on its accuracy, i.e. its ability to correctly allocate subjects to a certain prognosis or diagnosis. The ROC plot indicates the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of thresholds suitable for making a distinction. On the y-axis is sensitivity, or the true-positive fraction, which is defined as the ratio of number of true-positive test results to the product of number of true-positive and number of false-negative test results. It is calculated solely from the affected subgroup. On the x-axis is the false-positive fraction, or 1 - specificity, which is defined as the ratio of number of false-positive results to the product of number of true-negative and number of false-positive results. It is an index of specificity and is calculated entirely from the unaffected subgroup. Because the true- and false-positive fractions are calculated entirely separately, by using the test results from two different subgroups, the ROC plot is independent of the prevalence of the event in the cohort. Each point on the ROC plot represents a sensitivity / 1 - specificity pair corresponding to a particular decision threshold. A test with perfect discrimination (no overlap in the two distributions of results) has an ROC plot that passes through the upper left corner, where the true-positive fraction is 1.0, or 100% (perfect sensitivity), and the falsepositive fraction is 0 (perfect specificity). The theoretical plot for a test with no discrimination (identical distributions of results for the two groups) is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes. If the ROC plot falls completely below the 45° diagonal, this is easily remedied by reversing the criterion for "positivity" from "greater than" to "less than" or vice versa. Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test. Dependent on a desired confidence interval, a threshold can be derived from the ROC curve allowing for the diagnosis for a given event with a proper balance of sensitivity and specificity, respectively. Accordingly, the reference to be used for the method of the present invention, i.e. a threshold which allows the respective assessment, such as in the diagnosis of heart muscle tissue damage, the determination of the extent of heart failure tissue damage, and / or the diagnosis of atrial arrhythmia, can be generated, preferably, by establishing a ROC for said cohort as described above and deriving a threshold amount therefrom. Dependent on a desired sensitivity and specificity for the assessment, the ROC plot allows deriving a suitable threshold. It will be understood that an optimal sensitivity is desired for e.g. excluding a subject suffering from heart muscle tissue damage or atrial arrhythmia (i.e. a rule out) whereas an optimal specificity is envisaged for a subject to be diagnosed to suffer from heart muscle tissue damage (i.e. a rule in).

[0125] Since it is known in the art that the amount of RET decreases with age, an age-specific, i.e. age-matched, reference amount may be used. This is taken into account by the skilled person.

[0126] Preferably, the term “reference amount” herein refers to a predetermined value. Said predetermined value shall allow for the assessment as referred to herein, such as in the diagnosis of heart muscle tissue damage, the determination of the extent of heart failure tissue damage, the diagnosis of atrial arrhythmia and / or the assessment of mitral insufficiency.

[0127] In the method for the diagnosis of heart muscle tissue damage, for example, the reference, i.e. the reference amount shall allow for differentiating between a subject who suffers from such damage and a subject who does not suffer from such damage. In the method for the diagnosis of AF, for example, the reference shall allow for differentiating between a subject suffering from AF and a subject who is not suffering AF. In a method for assessing mitral insufficiency, the reference shall allow for differentiating between no mitral insufficiency or mitral insufficiency or between any one of no, Grad I, Grad II or Grad III mitral insufficiency.

[0128] In connection with the diagnosis of heart muscle tissue damage, the diagnostic algorithm is preferably as follows:

[0129] Preferably, an amount of the biomarker MyL7 and optionally, of the at least one further biomarker higher than the reference is indicative for a subject who suffers from heart muscle tissue damage, and / or an amount of the biomarker MyL7 and optionally, of the at least one further biomarker lower than the reference is indicative for a subject who does not suffer from heart muscle tissue damage.

[0130] In connection with the diagnosis of atrial arrhythmia, the diagnostic algorithm is preferably as follows:

[0131] Preferably, an amount of the biomarker MyL7 and optionally, of the at least one further biomarker higher than the reference is indicative for a subject who suffers from atrial a, and / or an amount of the biomarker MyL7 and optionally, of the at least one further biomarker lower than the reference is indicative for a subject who does not suffer from atrial arrhythmia.

[0132] In connection with the assessment of mitral insufficiency, the diagnostic algorithm is preferably as follows:

[0133] The reference amount may be also the amount of the biomarker in a sample that was obtained from a patient known to exhibit mitral insufficiency or mitral insufficiency of a predetermined degree (Grad I to III). An amount of the biomarker determined in a test sample that is identical or above the reference shall be indicative for the presence of mitral insufficiency or for a certain degree thereof.

[0134] Moreover, the reference amount may depend on the time point at which the sample has been taken (e.g. after surgery). This is taken into account by the skilled person.

[0135] The comparison step may comprise the calculation of a ratio of the determined amount(s) of the biomarker. For example, the ratio of the amount in the test sample to the reference amount (for example the amount in the first sample) is calculated. Based on the ratio, the heart muscle tissue damage is assessed. For example, when the ratio is in a range of 1.5 to 200.000 the amount of the biomarker in the patient is defined as increased compared to the reference amount. In various embodiments, an “increased amount” refers to an at least 1.5- fold increase, preferably an at least 2-fold increase, relative to the reference amount. Preferably, an “increased amount” refers to an at least 100-fold increase, in particular to an at least 1000-fold increase, relative to the reference amount. In an embodiment, the increase is an at least 10.000-fold increase, or even at least 50.000-fold increase relative to the reference amount.

[0136] The present invention further relates to a method of monitoring a cardiac surgery, comprising a) determining the amount of the biomarker MyL7 in a first sample obtained from a subject prior to a cardiac surgery, b) determining the amount of the biomarker MyL7 in a second sample obtained from a subject within 12 hours after said cardiac surgery, c) comparing the amount in the second sample to the amount in the first sample.

[0137] In an embodiment of the method of the present invention, the method further comprises the step of subjecting the patient to said cardiac surgery.

[0138] The present invention also relates to the use of the biomarker MyL7 (Myosin light chain 7) or of at least one detection agent which specifically binds to said biomarker in at least one sample from a subject for assessing mitral insufficiency.

[0139] Moreover, the present invention provides for a method for assessing mitral insufficiency in a subject comprising a) determining the amount of MyL7 in a sample of a subject suspected to suffer from mitral insufficiency; b) comparing the determined amount of MyL7 to a reference; and c) assessing mitral insufficiency based on said comparison.

[0140] Preferably, said assessing comprises diagnosing whether a subject suffers from mitral insufficiency, or not. More preferably, said assessing comprises differentiating between no mitral insufficiency, Grad I mitral insufficiency, Grad II mitral insufficiency and Grad III mitral insufficiency.

[0141] Moreover, the present invention relates to the in vitro use of the biomarker MyL7 (Myosin regulatory light chain 2, atrial isoform) or of at least one detection agent which specifically binds to said biomarker in a) a sample from a subject obtained prior to a surgery, and b) a sample from a subject obtained within 12 hour after surgery, for monitoring a cardiac surgery.

[0142] The term “monitoring a cardiac surgery” as used herein, preferably, refers to assessing the heart muscle tissue damage (preferably, atrial heart muscle tissue damage) caused by said surgery. More preferably, the term refers to “monitoring the success of the cardiac surgery”, in particular the effectiveness, such as the technical success of a cardiac surgery (in particular of an ablation). Thus, it is assessed whether the surgery, in particular an ablation, has been successful. A subject who has undergone a successful cardiac surgery is at lower risk of suffering from recurrence of atrial fibrillation (as compared to the average risk after cardiac surgery). A subject who has undergone an unsuccessful cardiac surgery is at higher risk of suffering from a recurrence of atrial fibrillation (as compared to the average risk after cardiac surgery). Such subjects are preferably closely monitored. Further, the ablation procedure might be repeated.

[0143] The term “subject”, “cardiac surgery” have been defined above. The definitions apply accordingly. In a preferred embodiment of the aforementioned method, the subject to be tested suffers from atrial fibrillation and the cardiac surgery is cardiac ablation. More preferably, the cardiac surgery is endocardial ablation or epicardial ablation. Alternatively, the subject suffers from atrial flutter or atrial tachycardia and the cardiac surgery is cardiac ablation.

[0144] Endocardial ablation or epicardial ablation are well-known in the art and described e.g. in Parameswaran et al. (Nat Rev Cardiol 18, 210-225 (2021)). Ablation lines are set in the heart atrium following a specific scheme to prevent the transmission of electrical impulses that cause the atria to fibrillate rather than contract in a regular pattern. This ablation can be performed as endocardial ablation after opening of the right and left atrium or as epicardial ablation of the pulmonary veins as a concomitant procedure. Typically, the endocardial ablation is endocardial cryoablation. Typically, the epicardial ablation is radiofrequency ablation. Endocardial ablation is a more aggressive form of ablation as epicardial ablation and leads to stronger heart muscle damage (see EP 3 775 914 Al).

[0145] The first sample serves as a reference sample. It shall have been obtained prior to surgery. Preferably, the first sample has been obtained within one week prior to the surgery, more preferably within three days prior to the surgery and most preferably within one day prior to the surgery. The second sample shall have been obtained within 12 hours after surgery, more preferably within 8 hours and even more preferably within 4 hours after surgery, and most preferably, within 3 hours after surgery.

[0146] In another preferred embodiment, the second sample has been obtained at least one hour, but no later than 8 hours after cardiac surgery. In yet another preferred embodiment, the sample has been obtained at least one hour, but no later than 4 hours after cardiac surgery. In yet another preferred embodiment, the sample has been obtained at least one hour, but no later than 3 hours after cardiac surgery.

[0147] Further, it is envisaged that the second sample has been obtained at a predetermined time after cardiac surgery. For example, the sample can be obtained, about one hour, at least two hours, at least three hours, at least four hours, at least five hours, at least six hours, at least seven hours or at least eight hours after completion of surgery.

[0148] In a preferred embodiment, the second sample has been obtained about 2 hours after surgery.

[0149] In an embodiment, the completion of surgery is at the time point at which the subject is admitted to the ICU (intensive care unit), i.e. at which the patient arrives as the ICU.

[0150] Preferably, an increase and, more preferably, a significant increase, and, most preferably, a statistically significant increase of the amount of the biomarker in the second sample as compared to the amount of the biomarker in the first sample indicates atrial heart muscle tissue damage caused by the surgery.

[0151] Preferably, an increase and, more preferably, a significant increase, and, most preferably, a statistically significant increase of the amount of the biomarker in the second sample as compared to the amount of the biomarker in the first sample indicates that the cardiac surgery has been successful.

[0152] Preferably, an “increase” which is a significant increase refers to an at least 100-fold increase, in particular to an at least 1000-fold increase, relative to the reference amount. In an embodiment, the increase is an at least 10.000-fold increase, or even at least 50.000-fold increase relative to the reference amount.

[0153] In case, the increase is lower than indicated above, e.g. lower than a 100-fold increase, the cardiac surgery was not successful. In this case, the subject might be closely monitored. Alternatively, the subject is subjection to the ablation procedure again. The present invention further relates to a method for determining the amount of the biomarker MyL7, and optionally the amount of at least one further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC, said method comprising a) providing at least one sample from a subject, wherein said sample has been obtained from said subject within 12 hours after cardiac surgery, and b) determining, in the at least one sample provided in step a), the amount of the biomarker MyL7 and, optionally, the amount of at least one further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC.

[0154] The present invention further relates to the use (preferably in vitro) of the biomarker MyL7 (Myosin regulatory light chain 2, atrial isoform) or of at least one detection agent which specifically binds to said biomarker in at least one sample from a subject for assessing heart muscle tissue damage. In a preferred embodiment of this use, the use further comprises the use of i) a cardiac Troponin or a detection agent which specifically binds a cardiac Troponin and / or of ii) cMyBPC or a detection agent which specifically binds cMyBPC.

[0155] The present invention further relates to the use (preferably in vitro) of at least one detection agent which specifically binds to MyL7 for determining the amount of MyL7 in a sample that has been obtained from a subject within 12 hours after cardiac surgery. In a preferred embodiment of this use, the use further comprises the use of i) a cardiac Troponin or a detection agent which specifically binds a cardiac Troponin for determining the amount of a cardiac Troponin and / or of ii) cMyBPC or a detection agent which specifically binds cMyBPC for determining the amount of cMyBPC.

[0156] The present invention further relates to a kit for assessing heart muscle tissue damage, said kit comprising at least one detection agent which specifically binds to MyL7 and optionally at least one detection agent which specifically binds to a cardiac Troponin and / or at least one detection agent which specifically binds to cMyBPC. Typically, the kit is adapted for carrying out the method of the present invention. Typically, the kit further comprises an instruction manual in order to carry out the method.

[0157] The term “kit” as used herein refers to a collection of the aforementioned components, preferably, provided separately or within a single container. The container also comprises instructions for carrying out the method of the present invention. These instructions may be in the form of a manual or may be provided by a computer program code which is capable of carrying out the comparisons referred to in the methods of the present invention and to establish an assessment accordingly when implemented on a computer or a data processing device. Further, the kit shall comprise at least one standard for a reference as defined herein above, i.e. a solution with a pre-defined amount for the biomarker(s) as referred to herein representing a reference amount.

[0158] In some embodiments, a kit disclosed herein includes at least one component or a packaged combination of components for practicing a disclosed method. By “packaged combination” it is meant that the kits provide a single package that contains a combination of one or more components, such as probes (for example, an antibody or antigen binding fragment thereof), controls, buffers, reagents (for example, conjugate and / or substrate), instructions, and the like, as disclosed herein. A kit containing a single container is also included within the definition of “packaged combination.” In some embodiments, the kits include at least one probe, for example an antibody (having specific affinity for an epitope of a biomarker as disclosed herein). For example, the kits may include an antibody that is labelled with a fluorophore or an antibody that is a member of a fusion protein. In the kit, the probe may be immobilized, and may be immobilized in a specific conformation. For example, an immobilized probe may be provided in a kit to specifically bind target protein, to detect target protein in a sample, and / or to remove target protein from a sample.

[0159] A container of the kits may be any container that is suitable for packaging and / or containing one or more components disclosed herein, including for example probes (for example, an antibody), controls, buffers, and reagents (for example, conjugate and / or substrate). Suitable materials include, but are not limited to, glass, plastic, cardboard or other paper product, wood, metal, and any alloy thereof. In some embodiments, the container may completely encase an immobilized probe(s) or may simply cover the probe to minimize contamination by dust, oils, etc., and exposure to light. In some further embodiments, he kits may comprise a single container or multiple containers, and where multiple containers are present, each container may be the same as all other containers, different than others, or different than some but not all other containers.

[0160] It is to be understood that as used in the specification and in the claims, “a” or “an” can mean one or more, depending upon the context in which it is used. Thus, for example, reference to “an” item can mean that at least one item can be utilized.

[0161] As used herein, the terms “have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements. The term “comprising” also encompasses embodiments where only the items referred to are present, i.e. it has a limiting meaning in the sense of “consisting of’.

[0162] Further, as used herein, the terms "particularly", "more particularly", “typically”, and “more typically” or similar terms are used in conjunction with additional / alternative features, without restricting alternative possibilities. Thus, features introduced by these terms are additional / alternative features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative features. Similarly, features introduced by "in an embodiment of the invention" or similar expressions are intended to be additional / alternative features, without any restriction regarding alternative embodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such way with other additional / alternative or non- additional / alternative features of the invention.

[0163] Further, it will be understood that the term “at least one” as used herein means that one or more of the items referred to following the term may be used in accordance with the invention. For example, if the term indicates that at least one sampling unit shall be used this may be understood as one sampling unit or more than one sampling units, i.e. two, three, four, five or any other number. Depending on the item the term refers to, the skilled person understands as to what upper limit the term may refer, if any.

[0164] The term “about” as used herein means that with respect to any number recited after said term an interval accuracy exists within in which a technical effect can be achieved. Accordingly, "about" as referred to herein, preferably, refers to the precise numerical value or a range around said precise numerical value of ±20 %, preferably ±15 %, more preferably ±10 %, or even more preferably ±5 %.

[0165] All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

[0166] Embodiments of the present inventions

[0167] The following embodiments are preferred embodiments of the invention. The definitions provided herein above apply mutatis mutandis. Use of the biomarker MyL7 (Myosin light chain 7) or of at least one detection agent which specifically binds to said biomarker in at least one sample from a subject for assessing heart muscle tissue damage. The use of embodiment 1, wherein the sample is a blood, serum or plasma sample or interstitial fluid. The use of embodiment 1 or 2, wherein the subject is a human subject. The use of any one of embodiments 1 to 3, wherein the detection agent is an antibody or antigen-binding fragment thereof. The use of any one of embodiments 1 to 4, wherein the subject is suspected to suffer from heart muscle tissue damage. The use of any one of embodiments 1 to 5, wherein the heart muscle tissue damage is atrial heart muscle damage. The use of any one of embodiments 1 to 6, wherein the sample has been obtained after cardiac surgery, in particular after cardiac surgery involving the atria of the heart. The use of embodiment 7, wherein the cardiac surgery is cardiac ablation (such as endocardial or epicardial ablation), left atrial appendage closure, or interatrial shunt closure. The use of embodiment 7 or 8, wherein the sample has been obtained within 12 hours, such as within in 8 hours, e.g. within 4 hours after surgery. The use of any one of the preceding embodiments, wherein the assessment of heart muscle tissue damage is selected from a) the diagnosis of heart muscle tissue damage, b) the differentiation between atrial and ventricular heart muscle tissue damage, c) the determination of the extent of heart muscle tissue damage, and / or d) the diagnosis of atrial arrhythmia. 11. The use of any one of the preceding embodiments further comprising the use of i) a cardiac Troponin or a detection agent which specifically binds a cardiac Troponin and / or of ii) cMyBPC or a detection agent which specifically binds cMyBPC.

[0168] 12. A method for assessing heart muscle tissue damage, said method comprising the determination of the amount of the biomarker MyL7 in at least one sample from a subject, and the assessment of heart muscle tissue damage based on the amount of said biomarker.

[0169] 13. The method of embodiment 12, wherein the sample has been obtained after cardiac surgery.

[0170] 14. The method of embodiment 12 or 13, further comprising the comparison of the amount of the biomarker to a reference amount.

[0171] 15. The method of embodiment 14, wherein the reference amount is a predetermined value.

[0172] 16. The method of embodiment 13 and 14, wherein the reference amount is amount of the biomarker in a sample from the subject obtained prior to cardiac surgery.

[0173] 17. The method of any one of the preceding embodiments further comprising the determination of the amount of a cardiac Troponin and / or the amount of cMyBPC.

[0174] 18. A method of assessing heart muscle tissue damage, said method comprising the steps of a) providing at least one sample from a subject, b) determining, in the at least one sample provided in step a), the amount of the biomarker MyL7 and, optionally, the amount of at least one further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC, and c) providing information on the determined amount of the biomarker MyL7 and optionally on the determined amount of the at least one further biomarker to a physician, thereby aiding in the assessment of heart muscle tissue damage.

[0175] 19. A method assessing heart muscle tissue damage, comprising: a) providing an assay for the biomarker MyL7 and, optionally, at least one further assay for a further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC, and b) providing instructions for using of assay results obtained or obtainable by said assay(s) in the assessment of heart muscle tissue damage. A computer-implemented method for assessing heart muscle tissue damage, comprising a) receiving, at a processing unit, a value for the amount of MyL7, and, optionally, at least one further value for the amount of at least one further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC, wherein said amount of MyL7 and, optionally, the amount of the at least one further biomarker have been determined in at least one sample from a subject, b) comparing, by said processing unit, the value or values received in step (a) to a reference or to references, and c) assessing heart muscle tissue damage based in the comparison step b). The method of any one of the preceding embodiments, further comprising the recommendation or the initiation of a suitable therapy. A method of monitoring a cardiac surgery, comprising a) determining the amount of the biomarker MyL7 in a first sample obtained from a subject prior to a cardiac surgery, b) determining the amount of the biomarker MyL7 in a second sample obtained from a subject within 12 hours after said cardiac surgery, c) comparing the amount in the second sample to the amount in the first sample. A method for diagnosing atrial fibrillation, said method comprising a) determining the amount of the biomarker MyL7 in at least one sample from a subject, b) comparing the thus determined amount to a suitable reference amount, and c) diagnosing atrial fibrillation in the subject based on the results of the comparison step. 24. The method of embodiment 23, wherein said diagnosing comprises differentiating between sinus arrhythmia, paroxysmal atrial fibrillation and persistent or permanent atrial fibrillation.

[0176] 25. The method of embodiment 24, wherein said differentiating is differentiating between sinus arrhythmia or paroxysmal atrial fibrillation on one hand and persistent or permanent atrial fibrillation on the other hand.

[0177] 26. Use of the biomarker MyL7 (Myosin light chain 7) or of at least one detection agent which specifically binds to said biomarker in at least one sample from a subject for assessing mitral insufficiency.

[0178] 27 A method for assessing mitral insufficiency in a subject comprising a) determining the amount of MyL7 in a sample of a subject suspected to suffer from mitral insufficiency; b) comparing the determined amount of MyL7 to a reference and c) assessing mitral insufficiency based on said comparison.

[0179] 28. The use of embodiment 26 or the method of embodiment 27, wherein said assessing comprises diagnosing whether a subject suffers from mitral insufficiency, or not.

[0180] 29. The use of embodiment 26 or the method of embodiment 27, wherein said assessing comprises differentiating between no mitral insufficiency, Grad I mitral insufficiency, Grad II mitral insufficiency and Grad III mitral insufficiency.

[0181] 30. A kit for assessing heart muscle tissue damage, said kit comprising at least one detection agent which specifically binds to MyL7 and optionally at least one detection agent which specifically binds to a cardiac Troponin and / or at least one detection agent which specifically binds to cMyBPC.

[0182] 31. Use of at least one detection agent which specifically binds to MyL7 for determining the amount of MyL7 in a sample that has been obtained from a subject within 12 hours after cardiac surgery.

[0183] 32. A method for determining the amount of the biomarker MyL7, and optionally the amount of at least one further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC, said method comprising a) providing at least one sample from a subject, wherein said sample has been obtained from said subject within 12 hours after cardiac surgery, and b) determining, in the at least one sample provided in step a), the amount of the biomarker MyL7 and, optionally, the amount of at least one further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC.

[0184] EXAMPLES

[0185] The invention will be merely illustrated by the following Examples. The said Examples shall, whatsoever, not be construed in a manner limiting the scope of the invention.

[0186] Example 1

[0187] A robust prototype assay (RPA) was developed for the biomarker MyL7 with monoclonal antibodies and evaluated in several cohorts (see Example 2 to 4). Cardiac Troponin T and cMyBPC were assessed as reference biomarkers that reflect overall cardiac muscle damage.

[0188] Example 2: MAPPING cohort

[0189] MyL7, cMyBPC and TnThs levels were determined in plasma samples of n=60 patients with blood sampled and biomarkers assayed before open chest surgery because of CABG or valve surgery. Evidence of different types of AF, paroxysmal AF (n=14) and persistent AF (n=16) or SR (controls, n=30) was generated during surgery with simultaneous Endo-Epicardial High Density Activation Mapping.

[0190] Epicardial High Density Mapping is a different means for discrimination among subgroups of sinus rhythm, paroxysmal and persistent AF according to different conduction patterns of fibrillation waves resulting from electric activations (see Eckstein et al., Transmural Conduction Is the Predominant Mechanism of Breakthrough During Atrial Fibrillation Evidence From Simultaneous Endo-Epicardial High-Density Activation Mapping. Circ Ar- rhythm Electrophysiol. (2013) 6, pp. 334-341; van Marion et al., Diagnosis and Therapy of Atrial Fibrillation: the Past, the Present and the Future Diagnosis and Therapy of Atrial Fibrillation: the Past, the Present and the Future JAFIB: Journal of Atrial Fibrillation;Aug / Sep2015, Vol. 8 Issue 2, p5).

[0191] The results are shown in Figure 1.

[0192] Data evaluation showed that patients with MyL7 levels above a reference value are suspected to have atrial fibrillation (AF). The observed AUC of MyL7 for the detection of paroxysmal AF or persistent AF was 0.73. In contrast the observed AUC of cMyBPC for the detection of paroxysmal AF or persistent AF was 0.55. Thus, in the patients analyzed herein, the biomarker MyL7 allows for an improved detection of AF versus cMyBPC. Thus, in the patients analyzed herein, MyL7 clearly overperforms cMyBPC. The evaluation of MyL7 in the MAPPING study demonstrates that modestly elevated circulating MyL7 titers are associated with persistent AF, a disease related to modest atrial muscle tissue damage.

[0193] The evaluation of MyL7 in the MAPPING study demonstrates detection of circulating atrial muscle protein MyL7 or fragments thereof in a disease related to spontaneous atrial muscle damage.

[0194] Example 3: MAZE cohort

[0195] MyL7, cMyBPC and TnThs levels have been determined in plasma samples of n=52 patients undergoing open chest surgery enrolled with serial blood sampling baseline, 2h, 4h, 6h and 24 hours afterwards. Patients underwent open chest surgery with epicardial or endocardial ablation (see Lahm et al. 2019, loc. cit). Upon artificially induced atrial damage, MyL7 is released into the blood with peak levels reached approximately two hours post-operation. The increase in MyL7 levels in the circulation correlates with the extent of the atrial damage (endocardial versus epicardial).

[0196] The results are shown in Figure 2.

[0197] Data evaluation in patients with endocardial ablation showed markedly elevated circulating levels after the intervention for all markers. However, the data clearly show a much higher relative change for MyL7 versus cMyBPC and TnThs.

[0198] MyBPHL, another cardiac biomarker with assumed atrial versus ventricular specificity was measured in samples of the MAZE cohort by the authors of Lahm et al. 2019. Published data in patients with endocardial ablation showed that MyBPHL values at admission to the ICU were observed to be about 3 to 4 times higher compared to the MyBPHL value prior to surgery.

[0199] MYL7 values at admission to the ICU were observed to be about 180000 times higher compared to the MyBPHL value prior to surgery. It is proposed that TnThs / Myl7 ratios or Myl7 alone may be used to assess severity of procedural related atrial muscle tissue damage.

[0200] The evaluation of MyL7 in the MAZE study demonstrates detection of circulating atrial muscle protein MyL7 or fragments thereof and the assessment of the severity of procedural induced atrial muscle tissue damage. Example 4: RRTAK cohort

[0201] MyL7, cMyBPC and TnThs levels were determined in plasma samples of n=51 patients undergoing percutaneous aortic valve replacement enrolled with serial blood sampling baseline, 4h, 8h, 24h, 48h, 72h, 1 week and 3 months afterwards.

[0202] It was observed that after percutaneous aortic valve replacement cardiac muscle proteins were only modestly elevated in the circulation. MyL7 levels in the blood declined more rapidly versus TnThs after the intervention. TnThs / Myl7 ratios may be useful to assess severity of procedural related atrial muscle tissue damage.

[0203] The evaluation of MyL7 in the RRTAK study is intended to demonstrate that pronounced elevations of MyL7 titers cannot be observed in patients undergoing cardiac interventions that are not specifically localized in the atrium.

[0204] Example 5: Increased MYL7 is an indicator for mitral insufficiency

[0205] Patients of the RRTAK were analyzed as described in Example 4 for MYL7 levels in blood samples. Mitral insufficiency is caused by a blood reflux into the left atrium that causes increased volume and pressure. Among the investigated patients of the RRTAK study, there were patients with different degrees of mitral insufficiency, i.e., no insufficiency, Grad I, Grad II and Grad III insufficiency.

[0206] The MYL7 levels found in the blood samples of the study participants correlated with the presence and with the degree of mitral insufficiency. Low levels were found in patients with no mitral insufficiency whereas highest levels were found in those with Grade II mitral insufficiency (Note: in light of the low number of participants exhibiting Grad III mitral insufficiency, statistics were not elusive). The results are shown in Fig. 3.

[0207] Example 6: Increased MYL7 in patients suffering from persisting atrial fibrillation compared to paroxysmal atrial fibrillation or sinus arrhythmia

[0208] Atrial fibrillation is associated with fibrotic remodeling of the left atrium. In a study (Mapping), MYL7 levels were determined in patients suffering from sinus arrhythmia, paroxysmal atrial fibrillation and persisting atrial fibrillation. High levels of MYL7 were found in patients suffering from persisting atrial fibrillation whereas the levels found in patients with sinus arrhythmia or paroxysmal atrial fibrillation were significantly lower. Results are shown in Fig. 4.

Claims

Claims1. Use of the biomarker MyL7 (Myosin light chain 7) or of at least one detection agent which specifically binds to said biomarker in at least one sample from a subject for assessing heart muscle tissue damage.

2. The use of claim 1, wherein the sample is a blood, serum or plasma sample or interstitial fluid.

3. The use of claim 1 or 2, wherein the subject is a human subject and / or wherein the subject is suspected to suffer from heart muscle tissue damage.

4. The use of any one of claims 1 to 3, wherein the heart muscle tissue damage is atrial heart muscle damage.

5. The use of any one of claims 1 to 4, wherein the sample has been obtained after cardiac surgery, in particular after cardiac surgery involving the atria of the heart, for example wherein the cardiac surgery is cardiac ablation (such as endocardial or epicardial ablation), left atrial appendage closure, or interatrial shunt closure.

6. The use of claim 5, wherein the sample has been obtained within 12 hours, such as within in 8 hours, e.g. within 4 hours after said cardiac surgery.

7. The use of any one of the preceding claims, wherein the assessment of heart muscle tissue damage is selected from a) the diagnosis of heart muscle tissue damage, b) the differentiation between atrial and ventricular heart muscle tissue damage, c) the determination of the extent of heart muscle tissue damage, and / or d) the diagnosis of atrial arrhythmia.

8. A method for assessing heart muscle tissue damage, said method comprising the determination of the amount of the biomarker MyL7 in at least one sample from a subject, and the assessment of heart muscle tissue damage based on the amount of said biomarker.

9. The method of claim 8, wherein a) the sample has been obtained after cardiac surgery, b) the method further comprises the comparison of the amount of the biomarker to a reference amount, in particular wherein the reference amount is a predetermined value, or wherein the reference amount is amount of the biomarker in a sample from the subject obtained prior to cardiac surgery.

10. A method of assessing heart muscle tissue damage, said method comprising the steps of a) providing at least one sample from a subject, b) determining, in the at least one sample provided in step a), the amount of the biomarker MyL7 and, optionally, the amount of at least one further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC, and c) providing information on the determined amount of the biomarker MyL7 and optionally on the determined amount of the at least one further biomarker to a physician, thereby aiding in the assessment of heart muscle tissue damage.

11. A method assessing heart muscle tissue damage, comprising: a) providing an assay for the biomarker MyL7 and, optionally, at least one further assay for a further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC, and b) providing instructions for using of assay results obtained or obtainable by said assay(s) in the assessment of heart muscle tissue damage.

12. A computer-implemented method for assessing heart muscle tissue damage, comprising a) receiving, at a processing unit, a value for the amount of MyL7, and, optionally, at least one further value for the amount of at least one further biomarker, said further biomarker being a cardiac Troponin and / or cMyBPC, wherein said amount of MyL7 and, optionally, the amount of the at least one further biomarker have been determined in at least one sample from a subject, b) comparing, by said processing unit, the value or values received in step (a) to a reference or to references, and c) assessing heart muscle tissue damage based in the comparison step b), comprising the recommendation or the initiation of a suitable therapy.

13. A method of monitoring a cardiac surgery, for example a cardiac ablation, comprising a) determining the amount of the biomarker MyL7 in a first sample obtained from a subject prior to a cardiac surgery, b) determining the amount of the biomarker MyL7 in a second sample obtained from a subject within 12 hours after said cardiac surgery, c) comparing the amount in the second sample to the amount in the first sample.

14. A method for diagnosing atrial fibrillation, said method comprising a) determining the amount of the biomarker MyL7 in at least one sample from a subject, b) comparing the thus determined amount to a suitable reference amount, and optionally c) diagnosing atrial fibrillation in the subject based on the results of the comparison step.

15. The method of claim 14, wherein said diagnosing comprises differentiating between sinus arrhythmia, paroxysmal atrial fibrillation and persistent or permanent atrial fibrillation.

16. The method of claim 14, wherein said differentiating is differentiating between sinus arrhythmia or paroxysmal atrial fibrillation on one hand and persistent or permanent atrial fibrillation on the other hand.

17. Use of the biomarker MyL7 (Myosin light chain 7) or of at least one detection agent which specifically binds to said biomarker in at least one sample from a subject for assessing mitral insufficiency.

18. A method for assessing mitral insufficiency in a subject comprising a) determining the amount of MyL7 in a sample of a subject suspected to suffer from mitral insufficiency; b) comparing the determined amount of MyL7 to a reference and c) assessing mitral insufficiency based on said comparison.

19. The use of claim 17 or the method of claim 18, wherein said assessing comprises diagnosing whether a subject suffers from mitral insufficiency, or not.

20. The use of claim 17 or the method of claim 18, wherein said assessing comprises differentiating between no mitral insufficiency, Grad I mitral insufficiency, Grad II mitral insufficiency and Grad III mitral insufficiency.

21. A kit for assessing heart muscle tissue damage, said kit comprising at least one detection agent which specifically binds to MyL7 and optionally at least one detection agent which specifically binds to a cardiac Troponin and / or at least one detection agent which specifically binds to cMyBPC.

22. Use of at least one detection agent which specifically binds to MyL7 for determining the amount of MyL7 in a sample that has been obtained from a subject within 12 hours after cardiac surgery, for example after a cardiac ablation.

Images