Use of myl7 or fragments thereof for assessing atrial myocardial tissue damage

Abstract

This invention relates to a method for assessing myocardial tissue damage. The method is based on the determination of the biomarker MyL7 in samples from a subject. Furthermore, this invention relates to the use of the biomarker MyL7 (myosin regulatory light chain 2, atrial subtype) or at least one detection agent for assessing myocardial tissue damage, said detection agent specifically binding to said biomarker in at least one sample from a subject. The invention further relates to a kit for assessing myocardial tissue damage.

Description

[0001] This invention relates to methods for assessing myocardial tissue damage. These methods are based on the determination of the biomarker MyL7 (myosin regulatory light chain 2, atrial subtype) in samples from subjects. Furthermore, this invention relates to the use of the biomarker MyL7 or at least one assay for assessing myocardial tissue damage, wherein the assay specifically binds to said biomarker in at least one sample from a subject. The invention further relates to a kit for assessing myocardial tissue damage. Background Technology

[0002] In developed countries, heart disease is a leading cause of morbidity and mortality. Currently, many biomarkers are used for the diagnosis of heart disease. For example, cardiac troponin and cardiac myosin-binding protein C (cMyBPC) can detect myocardial damage. However, neither of these biomarkers can differentiate between atrial and ventricular myocardial damage.

[0003] Ablation is a treatment for patients with atrial fibrillation, designed to correct the abnormal heart rhythm by blocking electrical pathways in the heart. Ablation can be performed during cardiac surgery. An ablation wire is placed within the atria of the heart to prevent the conduction of electrical impulses that cause atrial fibrillation. This ablation can be performed as either endocardial ablation or epicardial ablation. Both types of ablation damage myocardial tissue.

[0004] Biomarkers of atrial myocardial tissue damage are needed because they can be used to monitor the success of ablation therapy. Therefore, assessing the severity of atrial myocardial tissue damage in order to plan treatment accordingly is an important unmet need.

[0005] Furthermore, because cardiac troponin and myosin-binding protein C cannot differentiate between atrial and ventricular myocardial injury, it is impossible to determine whether the elevation of these biomarkers is caused by i) the ablation procedure or ii) additional myocardial injury to the ventricle. Currently, patients often undergo further diagnostic procedures, such as to rule out perioperative cardiac events.

[0006] EP 3 775 914 A1 discloses a method for diagnosing myocardial tissue damage (preferably atrial myocardial tissue damage) in patients based on the biomarker MyBPHL. Published data from patients undergoing endocardial ablation show that MyBPHL values ​​observed at ICU admission are approximately 3 to 4 times higher than preoperative MyBPHL values ​​(see Lahm, H., Dreßen, M., Beck, N. et al. Myosin binding protein H-like (MYBPHL): a promising biomarker to predict atrial damage. Sci Rep 9, 9986 (2019)).

[0007] Doll analyzed the proteome of the human heart by measuring 16 anatomical regions and three major cardiac cell types using high-resolution mass spectrometry-based proteomics (Doll, S., Dreßen, M., Geyer, PE et al. Region and cell-type resolved quantitative proteomic map of the human heart. Nat Commun 8, 1469 (2017)).

[0008] MyL7 ( m yosin l ight chain 7 Myosin light chain 7, also known as atrial light chain-2 (ALC-2) or myosin regulatory light chain 2, atrial subtype (MLC2a), is a protein encoded by the MYL7 gene in humans. It is expressed in the atria of the cardiac muscle in healthy individuals, where it plays a role in regulating cardiac development and contractility. MyL7 is known to be expressed in both atria (see Hailstones et al., Journal of Biological Chemistry, Vol. 267, No. 32, 1992, pp. 23295-23300).

[0009] Advantageously, the studies upon which this invention is based have shown that the biomarker MyL7 is a reliable marker of myocardial tissue damage, particularly atrial myocardial tissue damage. For example, studies have shown that this biomarker is elevated in patients with atrial fibrillation (a condition associated with mild atrial muscle tissue damage) (see Example 2 and Figure 1). Furthermore, this biomarker is also elevated after cardiac ablation (see Example 3 and Figure 2).

[0010] Contrary to published MyBPHL data, the inventors observed a significantly higher increase in MyL7 levels after cardiac ablation in the same scenario. Surprisingly, MyL7 levels observed at ICU admission were approximately 180,000 times higher than pre-operative MyL7 levels (compared to only about 3 to 4 times higher for MyBPHL). Therefore, this biomarker can reliably detect myocardial injury.

[0011] Interestingly, the research upon which this invention is based also demonstrates that the biomarker MyL7 can be used to determine the extent of myocardial tissue damage, particularly atrial myocardial tissue damage. Specifically, the study showed that the level of this biomarker was significantly higher after endocardial ablation than after epicardial ablation. Since endocardial ablation is known to cause more myocardial tissue damage than epicardial ablation, the biomarker MyL7 is correlated with the extent of myocardial tissue damage (see, for example, EP 3 775914 A1). Furthermore, this biomarker is correlated with cardiac troponin T (a recognized marker of myocardial injury) (see, for example, Figure 2A). Therefore, the higher the level of the biomarker MyL7, the greater the extent of myocardial damage (and vice versa). Thus, MyL7 can be advantageously used as a marker for assessing the extent of myocardial tissue damage. Attached Figure Description

[0012] Figure 1. Patient outcomes from the MAPPING study: A) MyL7 in patients with paroxysmal atrial fibrillation (parAF), persistent atrial fibrillation (persAF), and sinus rhythm (SR); B) MyL7 in patients with atrial fibrillation (AFib) and sinus rhythm (SR); C) ROC curve of the biomarker MyL7; D) MyBPC in patients with atrial fibrillation (AFib) and sinus rhythm (SR); E) ROC curve of the biomarker MyBPC.

[0013] Figure 2. Results of patients in the MAZE study: A) This figure shows the trend lines of normalized (relative to pre-ablation values, e.g., MYL7_normalized = (MYL7 at 2 h) / (MYL7 pre-ablation)) biomarkers over time for different ablation procedures. This figure is based on 52 patients: 32 who underwent endocardial (left and right) ablation, 6 who underwent endocardial (left) ablation, 13 who underwent epicardial (left and right) ablation, and 1 who underwent epicardial (right) ablation. The axes of all figures are fixed. In patients who underwent endocardial ablation, the increase in biomarker values ​​was much greater compared to those who underwent epicardial ablation; B) Same as Figure 2A, but with adjustable axes; C) Left and right ablation. Epicardium Examples of patients undergoing ablation. Normalized (relative to pre-OP) biomarker values ​​before ablation (before OP) and after ablation (2h-24h upon arrival at the ICU). Figure D: Left and right sides. Endocardium Examples of patients who underwent ablation. Normalized (relative to pre-OP) biomarker values ​​before ablation (before OP) and after ablation (2h-24h upon arrival at the ICU).

[0014] Figure 3. Patient outcomes from the RRTAK study. MYL7 levels correspond to the degree of mitral regurgitation. A total of 49 patients were enrolled. Nine patients did not show mitral regurgitation, 30 patients showed grade I mitral regurgitation, 8 patients showed grade II mitral regurgitation, and 2 patients showed grade III mitral regurgitation. Blood MYL7 levels correlated with the degree of mitral regurgitation, with low levels found in patients without mitral regurgitation, moderate levels found in grade I, and high levels found in grade II. The number of patients with grade III was too small.

[0015] Figure 4. Patient outcomes in the Mapping study. MYL7 levels correspond to persistent atrial fibrillation, paroxysmal atrial fibrillation, and sinus arrhythmia. A total of 46 patients were enrolled. 26 patients showed sinus arrhythmia and low MYL7 levels, 11 patients showed paroxysmal atrial fibrillation and low or slightly elevated MYL7 levels, and 9 patients showed persistent atrial fibrillation and elevated MYL7 levels. Summary of the Invention

[0016] The present invention relates to a method for assessing myocardial tissue damage, the method comprising determining the amount of a biomarker MyL7 in at least one sample from a subject, and assessing myocardial tissue damage based on the amount of said biomarker.

[0017] In a preferred embodiment of the method of the present invention, the method further includes comparing the amount of the biomarker with a reference amount.

[0018] The present invention further relates to a method for assessing myocardial tissue damage, the method comprising the following steps: a) Provide at least one sample from the subject, b) In at least one sample provided in step a), determine the amount of biomarker MyL7 and, optionally, the amount of at least one additional biomarker, said additional biomarker being cardiac troponin and / or cMyBPC, and c) Provide physicians with information about the amount of the identified biomarker MyL7 and, optionally, the amount of at least one other identified biomarker, to aid in the assessment of myocardial tissue damage.

[0019] The present invention further relates to a method for assessing myocardial tissue damage, the method comprising: a) Provides an assay for the biomarker MyL7 and, optionally, at least one additional assay for another biomarker, said other biomarker being cardiac troponin and / or cMyBPC, and b) Provide instructions on the use of the results obtained or available through the stated assay in the assessment of myocardial tissue damage.

[0020] The present invention further relates to a computer-implemented method for assessing myocardial tissue damage, the method comprising: a) Receive at the processing unit a value for the amount of MyL7 and, optionally, at least one additional value for the amount of at least one other biomarker, said additional biomarker being cardiac troponin and / or cMyBPC, wherein said amount of MyL7 and, optionally, the amount of at least one other biomarker, has been determined in at least one sample from the subject. b) The processing unit compares one or more values ​​received in step (a) with one or more reference values, and c) Assess myocardial tissue damage based on the comparison step b).

[0021] The present invention further relates to a method for monitoring cardiac surgery, the method comprising: a) Determine the amount of the biomarker MyL7 in the first sample obtained from the subject prior to cardiac surgery. b) Determine the amount of the biomarker MyL7 in a second sample obtained from the subject within 12 hours after the cardiac surgery. c) Compare the amount in the second sample with the amount in the first sample.

[0022] The present invention further relates to a method for determining the amount of the biomarker MyL7 and, optionally, the amount of at least one other biomarker, said other biomarker being cardiac troponin and / or cMyBPC, said method comprising: a) Provide at least one sample from the subject, wherein the sample was obtained from the subject within 12 hours after cardiac surgery, and b) In at least one sample provided in step a), determine the amount of biomarker MyL7 and optionally, the amount of at least one additional biomarker, said additional biomarker being cardiac troponin and / or cMyBPC.

[0023] The present invention further relates to the use of the biomarker MyL7 (myosin regulatory light chain 2, atrial subtype) or at least one detection agent for assessing myocardial tissue damage, the detection agent specifically binding to said biomarker in at least one sample from a subject. In a preferred embodiment of this use, the use further includes the use of: i) cardiac troponin or a detection agent specifically binding to cardiac troponin, and / or ii) cMyBPC or a detection agent specifically binding to cMyBPC.

[0024] The present invention further relates to the use of at least one detection agent that specifically binds to MyL7 for determining the amount of MyL7 in a sample obtained from a subject within 12 hours after cardiac surgery. In a preferred embodiment of this use, the use further includes the use of: i) cardiac troponin or a detection agent that specifically binds to cardiac troponin to determine the amount of cardiac troponin, and / or ii) cMyBPC or a detection agent that specifically binds to cMyBPC to determine the amount of cMyBPC.

[0025] The present invention further relates to a kit for assessing myocardial tissue damage, the kit comprising at least one detection agent that specifically binds to MyL7, and optionally at least one detection agent that specifically binds to cardiac troponin and / or at least one detection agent that specifically binds to cMyBPC.

[0026] In preferred embodiments of the methods, uses, or kits of the present invention, the assessment of myocardial tissue damage is selected from... a) Diagnosis of myocardial tissue injury, b) Differentiation between atrial and ventricular myocardial tissue injury. c) Determining the extent of myocardial tissue damage, and d) Diagnosis of atrial arrhythmias.

[0027] In a preferred embodiment of the method, use, or kit of the present invention, the subject is a human subject.

[0028] In a preferred embodiment of the method, use, or kit of the present invention, the subject is suspected of having myocardial tissue injury. Preferably, the myocardial tissue injury is atrial myocardial injury.

[0029] In a preferred embodiment of the present invention, the sample is blood, serum or plasma sample or interstitial fluid.

[0030] In a preferred embodiment of the invention, the sample is obtained after cardiac surgery, particularly cardiac surgery involving the atria of the heart. Preferably, the cardiac surgery is cardiac ablation (e.g., endocardial ablation or epicardial ablation), left atrial appendage occlusion, or atrial septal shunt occlusion. Preferably, the sample is obtained within 12 hours after the surgery, such as within 8 hours, for example, within 4 hours.

[0031] In a preferred embodiment of the method of the present invention, the method further includes the step of having the patient undergo cardiac surgery.

[0032] In a preferred embodiment of the present invention, the detection agent is an antibody or its antigen-binding fragment.

[0033] In a preferred embodiment of the present invention, cardiac troponin is cardiac troponin T or I. In particular, cardiac troponin is cardiac troponin T.

[0034] Detailed Implementation Methods / Definitions The present invention relates to a method for assessing myocardial tissue damage, the method comprising determining the amount of a biomarker MyL7 in at least one sample from a subject, and assessing myocardial tissue damage based on the amount of said biomarker in the at least one sample.

[0035] In a preferred embodiment of the method for assessing myocardial tissue damage, the method further includes a step of comparing the amount of the biomarker with a reference level of MyL7. Preferably, the assessment of myocardial tissue damage is based on the results of the comparison. Therefore, in embodiments, the method of the present invention includes the further step of assessing myocardial tissue damage in subjects based on the results of the comparison step.

[0036] Therefore, in embodiments, the present invention relates to a method for assessing myocardial tissue damage, the method comprising: a) Determine the amount of the biomarker MyL7 in at least one sample from the subject. b) Compare the quantity determined therefrom with a suitable reference quantity, and c) Assess myocardial tissue damage in subjects based on the results of the comparison step.

[0037] In embodiments, the method of the present invention may further include determining the amount of at least one additional biomarker, said additional biomarker being cardiac troponin and / or cMyBPC. The determined amount (or amounts) may be compared with a reference amount (or multiple reference amounts).

[0038] The methods referred to herein include methods that consist essentially of the steps mentioned or methods that include further steps. Furthermore, the methods of the present invention are preferably in vitro methods. In addition, these methods may include steps other than those explicitly mentioned. For example, further steps may involve determining additional biomarkers and / or sample pretreatment or evaluating the results obtained by the method. The methods of the present invention can also be used for monitoring, identifying, and subclassifying subjects. The methods can be performed manually or with automation assistance. Preferably, steps (a), (b), and / or (c) can be wholly or partially automated, for example, by means of suitable robots and sensory devices for the determination in step (a) or the computer-implemented calculations in step (b).

[0039] As those skilled in the art will understand, the assessments described herein are not typically intended to be accurate for all (i.e., 100%) of the subjects to be assessed. The term generally requires the ability to correctly assess a statistically significant subset of subjects (e.g., a cohort in a cohort study). Furthermore, actual assessments may include further steps, such as verification of the assessment. Therefore, in the context of this invention, the assessment assists physicians in assessing myocardial tissue damage. Thus, in the context of this invention, the term "assessment" preferably encompasses assisting physicians in assessing myocardial tissue damage.

[0040] Whether a subset is statistically significant can be determined by those skilled in the art using various well-known statistical assessment tools (e.g., confidence interval determination, p-value determination, Student's t-test, Mann-Whitney test, etc.) without further effort. See Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983 for details. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. Preferred p-values ​​are 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 in the population can be appropriately assessed by the method of the present invention.

[0041] The term "myocardial tissue injury" as used herein is well known in the art, see, for example, EP 3 775914 A1. As used herein, the term preferably refers to injury to myocardial tissue in the art. The term "injury" preferably includes damage and death (necrosis) of cells present in myocardial tissue.

[0042] The human heart comprises four chambers: the right atrium and left atrium, and the right ventricle and left ventricle. Therefore, the human heart has two atria and two ventricles. Preferably, the term "myocardial tissue injury" as used herein refers to atrial myocardial tissue injury, i.e., injury within the atria.

[0043] The term "subject" as used herein is preferably a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the subject is a human subject.

[0044] According to the present invention, the test subject is preferably a subject suspected of having myocardial tissue damage. In particular, the subject is a subject suspected of having atrial myocardial damage.

[0045] For example, subjects suspected of having myocardial tissue damage are preferably selected from the following: ● Subjects who underwent heart surgery, ● Subjects suspected of having an arrhythmia or who have an arrhythmia (preferably sinus arrhythmia or atrial fibrillation), ● Subjects suffering from cardiopulmonary congestion, ● Subjects with pulmonary embolism, ● Subjects with cardiomyopathy (such as atrial cardiomyopathy), and ● Subjects with pulmonary hypertension ● Subjects exhibiting mitral regurgitation.

[0046] In a preferred embodiment, the subject is a subject who has undergone cardiac surgery (preferably, cardiac surgery involving the atria). The term "cardiac surgery" is described elsewhere herein.

[0047] In another embodiment, the subject is a subject suspected of having an arrhythmia (preferably, atrial flutter or atrial fibrillation). In yet another embodiment, the subject is a subject with an arrhythmia (preferably, atrial flutter or atrial fibrillation). In yet another embodiment, the subject is a subject with pulmonary embolism. In yet another embodiment, the subject is a subject with cardiopulmonary congestion. In yet another embodiment, the subject is a subject with cardiomyopathy (preferably, atrial cardiomyopathy). In yet another embodiment, the subject is a subject with pulmonary hypertension.

[0048] In a preferred embodiment of the invention, the term "assessment of myocardial tissue damage" refers to... a) Diagnosis of myocardial tissue injury, b) Differentiation between atrial and ventricular myocardial tissue injury. c) Determining the extent of myocardial tissue damage, or d) Diagnosis of atrial arrhythmias.

[0049] Therefore, in embodiments of the present invention, this assessment is a diagnosis of myocardial tissue damage. Thus, it assesses whether the heart of the test subject shows myocardial tissue damage. In other words, it assesses whether these subjects suffer from myocardial tissue damage.

[0050] Therefore, the present invention relates to a method for diagnosing myocardial tissue injury, the method comprising: a) Determine the amount of the biomarker MyL7 in at least one sample from the subject. b) Compare the quantity determined therefrom with a suitable reference quantity, and c) Diagnose myocardial tissue damage in subjects based on the results of the comparison step.

[0051] Preferably, a level of the biomarker MyL7 above the reference level in the subject's sample indicates that the subject has myocardial tissue damage, while a level of the biomarker MyL7 below the reference level in the subject's sample indicates that the subject does not have myocardial tissue damage. Preferably, the myocardial tissue damage is atrial myocardial tissue damage.

[0052] In another preferred embodiment, the present invention relates to a method for differentiating between atrial myocardial tissue injury and ventricular myocardial tissue injury, the method comprising: a) Determine the amount of the biomarker MyL7 in at least one sample from the subject. b) Compare the quantity thus determined with a suitable reference quantity, and optionally... c) Based on the results of the comparison step, differentiate between atrial myocardial tissue injury and ventricular myocardial tissue injury.

[0053] According to the method described above for differentiating between atrial and ventricular myocardial tissue injury, the test subject preferably suffers from myocardial tissue injury. For example, the subject may have elevated blood levels of cardiac troponin and / or cMyBPC.

[0054] Preferably, a level of MyL7 above the reference level in the subject's sample indicates that the subject has atrial myocardial tissue damage, while a level of MyL7 below the reference level in the subject's sample indicates that the subject has ventricular tissue damage.

[0055] In another preferred embodiment, the present invention relates to a method for determining the extent of myocardial tissue damage, the method comprising: a) Determine the amount of the biomarker MyL7 in at least one sample from the subject, and b) Determine the extent of myocardial tissue damage in the subject based on the amount of biomarkers in at least one sample.

[0056] In embodiments, the extent of myocardial tissue damage (particularly atrial myocardial tissue damage) is determined in subjects who have undergone cardiac surgery, particularly those who have undergone cardiac ablation to treat arrhythmias (especially atrial fibrillation). Preferably, the subjects are human subjects, and the samples are blood, serum, or plasma samples. Preferred time points for obtaining samples after cardiac surgery are disclosed elsewhere herein. In step b), the extent of myocardial tissue damage is determined, for example, by comparing the amount of one or more markers with a reference amount (or multiple reference amounts). The extent is determined based on the result of the comparison. In embodiments, the reference amount is a predetermined value, such as the amount of a marker in a sample obtained from the subject before cardiac surgery. In some embodiments, the extent is determined by calculating the ratio of the amount of the marker after surgery to the amount of the marker before surgery.

[0057] In another preferred embodiment, the present invention relates to a method for diagnosing atrial fibrillation, the method comprising: a) Determine the amount of the biomarker MyL7 in at least one sample from the subject. b) Compare the quantity thus determined with a suitable reference quantity, and optionally... c) Diagnose atrial fibrillation in subjects based on the results of the comparison step.

[0058] The test subject associated with the above-described method for diagnosing atrial fibrillation is preferably a subject suspected of having atrial fibrillation (AF). Preferably, the subject suspected of having AF should have exhibited at least one symptom of AF before the method for diagnosing AF is performed (particularly before obtaining the sample). Symptoms of atrial fibrillation include dizziness, fainting, shortness of breath, and especially palpitations. In some embodiments, the subject has experienced at least one symptom within 12 hours (e.g., within 8 hours) before obtaining the test sample.

[0059] The atrial fibrillation to be diagnosed can be paroxysmal, permanent, or persistent. Preferably, the atrial fibrillation to be diagnosed is permanent or persistent.

[0060] Preferably, a level of MyL7 above the reference level in a subject's sample indicates that the subject has atrial fibrillation, while a level of MyL7 below the reference level in a subject's sample indicates that the subject does not have atrial fibrillation.

[0061] The terms “paroxysmal,” “persistent,” and “permanent” associated with atrial fibrillation are well-known in the field. The American College of Cardiology (ACC), the American Heart Association (AHA), and the European Society of Cardiology (ESC) have proposed the following classification system (see Fuster (2006) Circulation 114 (7): e257–354, the entire contents of which are incorporated herein by reference, see, for example, Figure 3 in the document): newly detected AF, paroxysmal AF, persistent AF, and permanent AF. All individuals with AF initially fall into the category referred to as newly detected AF. However, the subject may or may not have previously undetected episodes. If AF has persisted for more than one year, the subject has permanent AF. In particular, there is no conversion back to sinus rhythm (or only a conversion back to sinus rhythm with medical intervention). If AF has persisted for more than 7 days, the subject has persistent AF. The subject may require pharmacological or electrical intervention to terminate the atrial fibrillation. Therefore, persistent atrial fibrillation occurs during episodes, but the arrhythmia usually does not spontaneously (i.e., without medical inventions) revert to sinus rhythm. Paroxysmal atrial fibrillation preferably refers to intermittent episodes of atrial fibrillation that last no more than 7 days and terminate spontaneously (i.e., without medical intervention). In most cases of paroxysmal atrial fibrillation, episodes last less than 24 hours. Therefore, while paroxysmal atrial fibrillation terminates spontaneously, persistent atrial fibrillation does not terminate spontaneously and requires electrical or pharmacological cardioversion to terminate, or other surgical procedures such as ablation (Fuster (2006) Circulation 114 (7): e257-354). The term "sinus arrhythmia" as used herein refers to irregular changes in the heart's sinus rhythm. Preferably, the RR interval is greater than 0.12 s.

[0062] Preferably, as referred to herein, the diagnosis of atrial fibrillation means differentiating between sinus arrhythmias, paroxysmal atrial fibrillation, and persistent or permanent atrial fibrillation.

[0063] More preferably, the differentiation refers to the differentiation between sinus arrhythmia or paroxysmal atrial fibrillation on the one hand and persistent or permanent atrial fibrillation on the other hand.

[0064] As used herein, the term "sample" means any sample that contains the first, second, and / or third biomarkers described herein under physiological conditions. More typically, the sample is a bodily fluid sample, such as a blood sample or a sample derived therefrom (e.g., serum or plasma), a urine sample, interstitial fluid, a saliva sample, a lymph sample, etc. Most typically, the sample is a blood sample, a serum sample, or a plasma sample.

[0065] Furthermore, consider dried blood spots as the blood sample. A dried blood spot sample can be obtained by applying a drop of blood to absorbent filter paper. Allow the blood to fully saturate the filter paper and air dry for several hours. The blood may have been drawn from the subject using a scalpel, for example, from a finger.

[0066] 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 obtained after clotting. To obtain serum, blood clots are removed by centrifugation and the supernatant is collected. Plasma is the cell-free fluid fraction of blood. To obtain 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.

[0067] Blood samples include capillary blood samples. For example, such samples can be obtained from a puncture wound on a finger.

[0068] In a preferred embodiment of the invention, the sample is obtained after cardiac surgery. This allows for the assessment of whether the cardiac surgery has caused damage to myocardial tissue, specifically damage to part of the atrial myocardial tissue.

[0069] Heart surgery can be any type of surgery. In particular, heart surgery refers to heart surgery involving the atria of the heart.

[0070] In a preferred embodiment, the cardiac procedure is cardiac ablation. For example, the cardiac ablation is endocardial ablation. Alternatively, the cardiac ablation is epicardial ablation. Therefore, the test subject is a subject with atrial fibrillation who has undergone cardiac ablation. Alternatively, the test subject is a subject with atrial flutter or atrial tachycardia who has undergone cardiac ablation. Preferably, cardiac ablation has been performed to treat atrial fibrillation.

[0071] In another preferred embodiment, the cardiac procedure is a left atrial appendage occlusion. In yet another preferred embodiment, the cardiac procedure is an atrial septal shunt occlusion.

[0072] Preferably, the sample is obtained within 12 hours after the operation, more preferably within 8 hours after the operation, even more preferably within 4 hours after the operation, and most preferably within 3 hours after the operation.

[0073] In another preferred embodiment, the sample is obtained at least one hour but no later than eight hours after cardiac surgery. In yet another preferred embodiment, the sample is obtained at least one hour but no later than four hours after cardiac surgery. In still another preferred embodiment, the sample is obtained at least one hour but no later than three hours after cardiac surgery.

[0074] Furthermore, it is envisioned that the sample is obtained at a predetermined time after the heart surgery, i.e. at a fixed time (but within 12 hours after the surgery). For example, the sample may be obtained approximately 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 the surgery.

[0075] Furthermore, it is envisioned that more than one sample be obtained within 12 hours post-surgery. For example, it is envisioned that two, three, four, or more samples be obtained. This can be done to determine the time progression of the biomarker quantity. Preferably, these samples are obtained at intervals of at least 29 minutes, for example, at least 59 minutes.

[0076] The end of the surgery is typically considered the completion of the procedure. In this example, completion occurs at the point when the subject is admitted to the ICU.

[0077] A sample can also be obtained before surgery. As further explained below, this sample can be used as a reference sample.

[0078] According to the present invention, the amount of the biomarker MyL7 (myosin light chain 7) should be determined. MyL7 is a protein encoded by the MYL7 gene in humans. This protein is also known as atrial light chain-2 (ALC-2), myosin regulatory light chain 2, atrial subtype (MLC2a), or MyL2A. The protein has a molecular weight of about 19.4 kDa and contains 175 amino acids. It is an EF-chiral protein that binds to the neck region of the α-myosin heavy chain. The sequence of MyL7 is well known in the art. For example, it can be found under UniProt accession number Q01449 (MLRA_HUMAN). This biomarker is known to be expressed in both atria (see Hailstones et al., Journal of Biological Chemistry, Vol. 267, No. 32, 1992, pp. 23295-23300).

[0079] In various embodiments of the method described herein, the method further includes determining the level of at least one additional biomarker of myocardial tissue injury in at least one sample from a subject. Preferably, the at least one additional biomarker is cardiac troponin and / or cMyBPC. One or more additional biomarkers may be compared to a reference level (or multiple reference levels).

[0080] Preferably, an increase in one or more biomarkers compared to one or more reference levels indicates a subject with myocardial tissue damage (or atrial fibrillation). Preferably, a decrease in one or more biomarkers compared to one or more reference levels indicates a subject without myocardial tissue damage (or atrial fibrillation).

[0081] The term "cardiac troponin" refers to all troponin isoforms expressed in cardiac cells, and preferably in subendocardial cells. These isoforms are well characterized in the art, as described, for example, 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. Therefore, in the method of the present invention, both tropons can be identified together (i.e., simultaneously or sequentially) or individually (i.e., other isoforms are not identified at all). The 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" also covers variants of the aforementioned specific troponin, preferably variants of troponin T or troponin I.

[0082] In a preferred embodiment, the term "cardiac troponin" refers to cardiac troponin T (which is measured in the example). In another preferred embodiment, the term "cardiac troponin" refers to cardiac troponin I.

[0083] Myosin-binding protein C is a myosin-related protein located in the bridging region (C region) of the A band in skeletal muscle. According to the present invention, the amount of the cardiac isotype, namely cMyBPC (cardiac myosin-binding protein C) (also known as MYBPC3, CMD1MM, CMH4, FHC, LVNC10, MYBP-C, myosin-binding protein C, cardiac cMyBP-C, myosin-binding protein C3), should be determined. cMyBPC is produced in the myocardium. cMyBPC is encoded by the MYBPC3 gene. Further information on human cMYBPC can be found in the UniProtKB database, accession number Q14896 (MYPC3_HUMAN).

[0084] As used herein, the term “determine” refers to the qualitative and quantitative determination of a biomarker mentioned in the present invention, that is, the term covers the determination of the presence or absence of the biomarker or the determination of the absolute or relative amount of the biomarker. Preferably, “determining” the amount of a biomarker as referred to herein means the quantification of the biomarker, for example, measuring the level of the biomarker in a sample using appropriate detection methods described elsewhere herein. The terms “measure” and “determine” are used interchangeably herein.

[0085] Typically, the amount of the biomarker mentioned in this invention can be determined by immunoassay using sandwich, competitive, or other assay methods. The assay will produce a signal indicating the presence or absence, or the amount, of the biomarker.

[0086] Measuring the amount of a biomarker may preferably include the following steps: (a) contacting the peptide with a reagent that specifically binds the peptide; (b) (optionally) removing unbound reagent; and (c) measuring the amount of bound reagent, i.e., the reagent complex formed in step (a). According to a preferred embodiment, the contact, removal, and measurement steps may be performed by an analyzer unit. According to some embodiments, the steps may be performed by a single analyzer unit of the system or by more than one analyzer unit operatively communicating with each other. For example, according to a particular embodiment, the system disclosed herein may include a first analyzer unit for performing the contact and removal steps; and a second analyzer unit operatively connected to the first analyzer unit via a transmission unit (e.g., a robotic arm), which performs the measurement step.

[0087] A reagent that specifically binds to a biomarker (also referred to herein as a “binding reagent”) may be covalently or non-covalently coupled to a label that allows the detection and measurement of the bound reagent. Labeling can be performed by direct or indirect methods. Direct labeling involves directly (covalently or non-covalently) coupling a label to the binding reagent. Indirect labeling involves the binding (covalently or non-covalently) of a secondary binding reagent to a first binding reagent. The secondary binding reagent should specifically bind to the first binding reagent. The secondary binding reagent may be coupled to a suitable label and / or a target (receptor) of a tertiary binding reagent that binds to the secondary binding reagent. Suitable secondary and higher-order binding reagents may include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). The binding reagent or substrate may also be “labeled” with one or more labels as known in the art.

[0088] Such tags can then be targets for higher-order binding agents. Suitable tags include biotin, digitoxin, His tags, glutathione S-transferase, FLAG, GFP, myc tags, influenza A virus hemagglutinin (HA), maltose-binding protein, etc. In the case of peptides or polypeptides, the tag is preferably located at the N-terminus and / or C-terminus. Suitable tags are any tags detectable by suitable detection methods. Typical tags include gold particles, latex beads, acridinium ester, luminol, ruthenium complexes, iridium complexes, enzyme activity tags, radioactive tags, magnetic tags ("e.g., magnetic beads," including paramagnetic and superparamagnetic tags), and fluorescent tags. Enzyme activity tags include, for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, luciferase, and their derivatives. Suitable substrates for detection include diaminobenzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate, available as ready-made stock solutions from Roche Diagnostics), CDP-Star™ (Amersham Biosciences), and ECF™ (Amersham Biosciences). Suitable enzyme-substrate combinations can produce colored reaction products, fluorescence, or chemiluminescence, which can be measured using methods known in the art, such as the use of photographic film or a suitable camera system. The standards given above apply similarly for measuring enzyme reactions. Typical fluorescent labels include fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red, fluorescein, and Alexa dyes (e.g., Alexa 568). Additional fluorescent tags are available, for example, from MolecularProbes (Oregon). The use of quantum dots as fluorescent tags is also envisioned. Radioactive tags can be detected by any known and appropriate method, such as photographic film or phosphorescent imaging.

[0089] The amount of peptide can also preferably be measured as follows: (a) contacting a solid support containing a binding agent of the peptide as described elsewhere herein with a sample containing the peptide or polypeptide, and (b) measuring the amount of peptide or polypeptide bound to the support. Materials used to manufacture the support are well known in the art and include, in particular, commercial column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and / or silicon wafers and surfaces, nitrocellulose tapes, membranes, sheets, durable cells, pores and walls of reaction discs, plastic tubing, etc.

[0090] In another aspect, the sample is removed from the complex formed between the binding agent and at least one marker before the amount of the formed complex is measured. Thus, in one aspect, the binding agent may be immobilized on a solid support. In another aspect, the sample can be removed from the complex formed on the solid support by applying a washing solution.

[0091] Sandwich assays are among the most useful and commonly used assays, encompassing many variations of the sandwich assay technique. Simply put, 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 an appropriate incubation period, a second (detection) binding agent labeled with a reporter molecule capable of producing a detectable signal is added and incubated for a time sufficient to allow the formation of a binding agent-biomarker complex, thus allowing sufficient time for the formation of another complex of binding agent-biomarker-labeled binding agent. Any unreacted material can be washed away, and the presence of the biomarker is determined by observing the signal produced by the reporter molecule bound to the detection binding agent. The results can be qualitatively determined by simply observing the visible signal, or quantitatively determined by comparison with a control sample containing a known amount of the biomarker.

[0092] The incubation steps for typical sandwich assays can be varied as needed and as appropriate. Such variations include, for example, simultaneous incubation, where two or more binding reagents and biomarkers are co-incubated. For instance, the analyte and the labeled binding reagent are added simultaneously to a fixed capture binding reagent. Alternatively, the analyte and the labeled binding reagent may be incubated first, followed by the addition of an antibody that binds to or is capable of binding to the solid.

[0093] The complex formed between the specific binding reagent and the biomarker should be proportional to the amount of the biomarker present in the sample. It should be understood that the specificity and / or sensitivity of the binding reagent to be applied defines the proportion of at least one biomarker present in the sample that can be specifically bound. Further details on how this measurement can be performed can be found elsewhere in this document. The amount of the complex formed should be converted into the amount of the biomarker, thus reflecting the actual amount present in the sample.

[0094] The terms “binding reagent,” “specific binding reagent,” “analyte-specific binding reagent,” “detector,” and “reagent that specifically binds to a biomarker” are used interchangeably herein. Preferably, it refers to a reagent containing a binding portion that specifically binds to the corresponding biomarker. Examples of “binding reagent” or “reagent” are nucleic acid probes, nucleic acid primers, DNA molecules, RNA molecules, aptamers, antibodies, antibody fragments, peptides, peptide nucleic acids (PNAs), or compounds. Preferred reagents are antibodies or antigen-binding fragments thereof that specifically bind to the biomarker to be measured. The term “antibody” is used herein in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity (i.e., their antigen-binding fragments). Preferably, the antibody is a polyclonal antibody. More preferably, the antibody is a monoclonal antibody.

[0095] The term "specific binding" or "specifically binding" refers to a binding reaction in which the binding pair of molecules binds to each other under conditions where they do not bind significantly to other molecules. When referring to proteins or peptides as biomarkers, the term "specific binding" or "specifically binding" refers to a binding reaction in which the binding reagents are at least 10 -7 M has an affinity for the corresponding biomarker. The term "specific binding" or "specifically binding" preferably refers to an affinity of at least 10 for its target molecule. -8 M or even more preferably at least 10 -9 Affinity of M. The term "specific" or "specifically" is used to indicate that other molecules present in the sample do not significantly bind to the binding agent that is specific to the target molecule.

[0096] The determination of biomarkers as described herein may include mass spectrometry (MS) performed after a separation step, such as by LC or HPLC. As used herein, mass spectrometry encompasses all techniques that allow the determination of the molecular weight (i.e., mass) or mass variable corresponding to the compound (i.e., biomarker) to be determined according to the invention. Preferably, as used herein, mass spectrometry involves 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 combination of the above techniques. How to apply these techniques is well known to those skilled in the art. Furthermore, suitable apparatus is commercially available. More preferably, as used herein, mass spectrometry involves LC-MS and / or HPLC-MS, i.e., mass spectrometry operatively linked to a preceding 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 fragmentation occurring between stages. The mass spectrometer is coupled to a chromatographic apparatus. The sample, already separated by chromatography, is sorted and weighed in the first mass spectrometer, then fragmented in an inert gas collision cell, and one or more fragments are sorted and weighed in the second mass spectrometer. The fragments are then classified and weighed in the second mass spectrometer. Identification using MS / MS is more accurate.

[0097] In embodiments, the mass spectrometry method used herein encompasses quadrupole MS. Most preferably, the quadrupole MS is performed as follows: a) selecting the mass / charge quotient (m / z) of ions generated by ionization in a first analytical quadrupole of the mass spectrometer; b) fragmenting the ions selected in step a) by applying an accelerating voltage in a subsequent quadrupole filled with a collision gas and acting as a collision chamber; c) selecting the mass / charge quotient of ions generated by the fragmentation process in step b) in another subsequent quadrupole, whereby steps a) to c) of the method are performed at least once; and analyzing the mass / charge quotient of all ions present in the mixture due to the ionization process, whereby the quadrupole is filled with a collision gas, but no accelerating voltage is applied during the analysis. Details of the most preferred mass spectrometry method to be used according to the invention can be found in WO2003 / 073464.

[0098] More preferably, the mass spectrometry method is liquid chromatography (LC) MS, such as high performance liquid chromatography (HPLC) MS, especially HPLC-MS / MS. As used herein, liquid chromatography refers to all techniques that allow the separation of compounds (i.e., metabolites) in a liquid or supercritical phase.

[0099] In mass spectrometry, analytes in a sample are ionized to produce charged molecules or molecular fragments. The mass-to-charge ratio of the ionized analyte, particularly the ionized biomarker or its fragments, is then measured. Before ionization, the sample can be cleaved with a protease, such as trypsin. The protease breaks down protein biomarkers into smaller fragments.

[0100] Therefore, the mass spectrometry analysis step preferably includes an ionization step, in which the biomarker to be identified is ionized. Of course, other compounds present in the sample / elutions are also ionized. Ionization of biomarkers can be performed by any method deemed suitable, particularly by electron impact ionization, fast atom impact ionization, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and matrix-assisted laser desorption / ionization (MALDI).

[0101] In a preferred embodiment, the ionization step (for mass spectrometry) is performed by electrospray ionization (ESI). Therefore, the mass spectrometry is preferably ESI-MS (or, if tandem MS is performed: ESI-MS / MS). Electrospray ionization is a soft ionization method that can form ions without breaking any chemical bonds.

[0102] As used herein, the term "amount" means the absolute amount of a compound mentioned herein, the relative amount or concentration of the compound, and any value or parameter associated with or derived therefrom. Such values ​​or parameters include intensity signal values ​​from all specific physical or chemical properties obtained by direct measurement of the compound, such as intensity values ​​in mass spectrometry or NMR spectra. Furthermore, all values ​​or parameters obtained by indirect measurement specified elsewhere in this specification are included, for example, response levels determined from a bioreading system in response to an intensity signal obtained from the compound or from a specifically bound ligand. It should be understood that values ​​associated with the above-described amounts or parameters can also be obtained through all standard mathematical operations.

[0103] As used herein, the term "comparison" refers to comparing the amount of a biomarker in a sample from a subject with a reference amount of the biomarker specified elsewhere in this specification. It should be understood that, as used herein, comparison generally refers to a comparison of corresponding parameters or values, such as comparing an absolute amount with an absolute reference amount, or a concentration with a reference concentration, or comparing an intensity signal obtained from a biomarker in a sample with an intensity signal of the same type obtained from a reference sample. Comparisons can be performed manually or computer-assisted. Therefore, comparisons can be made by a computing device. For example, the value of a measured or detected amount of a biomarker in a sample from a subject can be compared to a reference amount, and such comparisons can be performed automatically by a computer program executing an algorithm for the comparison. The computer program performing the assessment will provide the desired rating in a suitable output format. For computer-assisted comparisons, the value of the measured amount can be compared to a value stored in a database by the computer program that corresponds to a suitable reference. The computer program can further evaluate the results of the comparison, i.e., automatically provide the desired rating in a suitable output format. For computer-assisted comparisons, the value of the measured amount can be compared to a value stored in a database by the computer program that corresponds to a suitable reference. Computer programs can further evaluate the results of the comparison, that is, automatically provide the expected assessment in an appropriate output format.

[0104] In a preferred embodiment of the invention, the assessment of myocardial tissue damage is a diagnosis of atrial myocardial tissue damage in a human subject, wherein the sample is a blood, serum, or plasma sample obtained after the subject has undergone cardiac ablation (particularly within 12 hours after the subject has undergone cardiac ablation). In another preferred embodiment of the invention, atrial myocardial tissue damage is diagnosed, wherein the sample is a blood, serum, or plasma sample obtained within 8 hours after the subject has undergone cardiac ablation. In yet another preferred embodiment of the invention, atrial myocardial tissue damage is diagnosed, wherein the sample is a blood, serum, or plasma sample obtained within 4 hours after the subject has undergone cardiac ablation. Preferably, cardiac ablation has been performed to treat atrial arrhythmias, particularly atrial fibrillation, in the subject.

[0105] In another preferred embodiment of the invention, the assessment of myocardial tissue damage involves determining the extent of atrial myocardial tissue damage in a human subject, wherein the sample is a blood, serum, or plasma sample obtained after the subject has undergone cardiac ablation, particularly within 12 hours after the ablation. In another preferred embodiment of the invention, the extent of atrial myocardial tissue damage is determined, wherein the sample is a blood, serum, or plasma sample obtained within 8 hours after the subject has undergone cardiac ablation. In yet another preferred embodiment of the invention, the extent of atrial myocardial tissue damage is determined, wherein the sample is a blood, serum, or plasma sample obtained within 4 hours after the subject has undergone cardiac ablation. Preferably, cardiac ablation has been performed to treat atrial arrhythmias, particularly atrial fibrillation.

[0106] In yet another preferred embodiment of the invention, the assessment of myocardial tissue damage is a diagnosis of atrial fibrillation in a subject suspected of having atrial fibrillation, wherein the sample is a blood, serum, or plasma sample.

[0107] The definitions and explanations provided above, with necessary modifications, apply to the following methods, uses, kits, and apparatus of this invention.

[0108] The present invention further relates to a method for assessing myocardial tissue damage, the method comprising the following steps: a) Provide at least one sample from the subject, b) In the at least one sample provided in step a), determine the amount of biomarker MyL7 and, optionally, the amount of at least one additional biomarker, said additional biomarker being cardiac troponin and / or cMyBPC, and c) Provide the physician with information about the amount of the identified biomarker MyL7 and, optionally, the amount of the identified at least one other biomarker, thereby aiding in the assessment of myocardial tissue damage.

[0109] Step a) of the method described above (providing at least one sample from the subject) does not involve extracting the sample from the subject. Preferably, the sample is obtained by receiving a sample from the subject. Therefore, the sample may have been delivered to a laboratory, for example, to determine one or more biomarkers.

[0110] The physician should be the attending physician, i.e., the physician who requests the determination of the biomarkers. The methods described above should assist the attending physician in assessing myocardial tissue damage. Therefore, this method does not cover the assessments mentioned above in conjunction with the methods for myocardial tissue damage. Information regarding the quantity of one or more biomarkers can be provided electronically.

[0111] The present invention further relates to a method for assessing myocardial tissue damage, the method comprising: a) Provides an assay for the biomarker MyL7 and, optionally, at least one additional assay for another biomarker, said other biomarker being cardiac troponin and / or cMyBPC, and b) Provide instructions on the use of the results obtained or available through the stated assay in this assessment of myocardial tissue damage.

[0112] These instructions should contain protocols for implementing the methods for assessing myocardial tissue damage as described above. Furthermore, these instructions should contain at least one reference value for the biomarker MyL7 and, optionally, at least one reference value for another biomarker, such as cardiac troponin and / or cMyBPC.

[0113] "Assay" preferably refers to a kit suitable for determining the amount of a biomarker. The term "kit" is explained below.

[0114] The measurement result obtained or available through the test is the value of the amount of one or more biomarkers.

[0115] The present invention further relates to a computer-implemented method for assessing myocardial tissue damage, the method comprising: a) Receive at the processing unit a value for the amount of MyL7 and, optionally, at least one additional value for the amount of at least one other biomarker, said additional biomarker being cardiac troponin and / or cMyBPC, wherein said amount of MyL7 and, optionally, the amount of at least one other biomarker, has been determined in at least one sample from the subject. b) The processing unit compares one or more values ​​received in step (a) with one or more reference values, and c) Assess myocardial tissue damage based on the comparison step b).

[0116] The above method is a computer-implemented method. Preferably, all steps of the computer-implemented method are executed by one or more processing units of a computer (or computer network). Therefore, the evaluation in step (c) is performed by the processing unit. Preferably, the evaluation is based on the result of step (b).

[0117] As described elsewhere in this document, one or more values ​​received in step (a) should be derived from determining the amount of biomarkers from the subject. Preferably, the value is a value representing the concentration of the biomarker. This value will typically be received by the processing unit by uploading or sending the value to the processing unit. Alternatively, the processing unit can receive the value by inputting the value via a user interface.

[0118] In an embodiment of the foregoing method, one or more reference values ​​proposed in step (b) are established from memory. Preferably, the reference values ​​are established from memory.

[0119] In an embodiment of the computer-implemented method of the present invention described above, the result of the evaluation performed in step c) is provided via a display configured to present the result.

[0120] In an embodiment of the computer-implemented method of the present invention described above, the method may include a further step of transmitting information regarding the assessment performed in step c) to the subject's electronic medical record.

[0121] In embodiments of methods for assessing myocardial tissue damage, these methods further include the step of recommending and / or initiating therapy for atrial fibrillation based on the assessment results. For example, if a subject is diagnosed with AF, therapy is recommended or initiated. For example, therapy with at least one anticoagulant may be recommended or initiated. In a preferred embodiment, the at least one anticoagulant is selected from the group consisting of heparin, coumarin derivatives (i.e., vitamin K antagonists) (especially warfarin or dicumarol), oral anticoagulants (especially dabigatran, rivaroxaban, or apixaban), tissue factor pathway inhibitors (TFPIs), antithrombin III, factor IXa inhibitors, factor Xa inhibitors, factor Va and factor VIIIa inhibitors, and thrombin inhibitors (anti-IIa type). Alternatively, if a subject is diagnosed with AF, cardiac ablation is recommended or initiated.

[0122] According to the invention, the amount of the biomarker MyL7 (and optionally, at least one other biomarker) should be compared with a reference, i.e., with a reference amount (or with multiple reference amounts). Therefore, the reference is preferably a reference amount. The terms "reference amount" or "reference" are well understood by those skilled in the art. It should be understood that the reference amount should allow for the assessment of myocardial tissue damage, such as in the diagnosis of myocardial tissue damage, the determination of the extent of myocardial tissue damage, and / or the diagnosis of atrial arrhythmias. For example, in conjunction with methods for diagnosing myocardial tissue damage, the reference amount preferably refers to an amount that allows subjects to be classified into either: (i) a group of subjects with myocardial tissue damage, or (ii) a group of subjects without myocardial tissue damage. For example, in conjunction with methods for diagnosing atrial arrhythmias, the reference amount preferably refers to an amount that allows subjects to be classified into either: (i) a group of subjects with atrial arrhythmias, and / or (ii) a group of subjects without atrial arrhythmias.

[0123] In principle, a reference value for the cohort of subjects specified above can be calculated by applying standard statistical methods based on the mean or average of a given biomarker. In particular, the accuracy of a test (such as a method designed to diagnose the occurrence or absence of an event) is best described by its receiver operating characteristic (ROC) (see, in particular, Zweig MH. et al., Clin. Chem. 1993;39:561-577). An ROC curve is a curve showing all pairs of sensitivity versus specificity produced by continuously varying the decision threshold across the entire observed data range. The clinical performance of a diagnostic method depends on its accuracy, i.e., its ability to correctly assign subjects to a given prognosis or diagnosis. The ROC curve shows the overlap between the two distributions by plotting the sensitivity versus 1-specificity across the entire range of thresholds applicable to the distinction. The y-axis represents sensitivity, i.e., the true positive score, which is defined as the ratio of the number of true positive test results to the product of the number of true positive test results and the number of false negative test results. It is calculated only from the affected subgroup. The x-axis represents the false positive score, or 1-specificity, which is defined as the ratio of the number of false positive results to the product of the number of true negative results and the number of false positive results. This is a specificity index and is calculated entirely from the unaffected subgroups. Because the true positive score and the false positive score are calculated completely separately, the ROC curve is independent of the incidence of the event in the cohort by using test results from two different subgroups. Each point on the ROC curve represents a sensitivity / 1-specificity pair corresponding to a specific decision threshold. Tests with perfect discrimination (no overlap in the two outcome distributions) have an ROC curve that crosses the top left corner, where the true positive score is 1.0 or 100% (perfect sensitivity) and the false positive score is 0 (perfect specificity). Tests with no discrimination (identical outcome distributions in both groups) have a theoretical curve that runs diagonally from the bottom left to the top right corner at a 45° angle. Most curves fall between these two extremes. If the ROC curve falls completely below the 45° diagonal, it can be easily corrected by reversing the "positive" criterion from "greater than" to "less than" or vice versa. Qualitatively, the closer the curve is to the upper left corner, the higher the overall accuracy of the test. Based on the desired confidence interval, a threshold can be derived from the ROC curve, allowing for diagnosis of a given event with an appropriate balance between sensitivity and specificity. Therefore, a reference for the method of the present invention (i.e., a threshold that allows for the corresponding assessment, such as in the diagnosis of myocardial tissue damage, the determination of the extent of myocardial tissue damage, and / or the diagnosis of atrial arrhythmias) can preferably be generated by establishing the ROC of the cohort as described above and thereby deriving the threshold amount. The ROC curve allows for the determination of an appropriate threshold based on the desired sensitivity and specificity of the assessment.It should be understood that optimal sensitivity is what is expected, for example, for excluding subjects with myocardial tissue damage or atrial arrhythmias (i.e., exclusion), while optimal specificity is envisioned for subjects diagnosed with myocardial tissue damage (i.e., confirmation).

[0124] Since it is known in the art that the amount of RET decreases with age, an age-specific, i.e., age-matched reference amount can be used. Those skilled in the art will take this into account.

[0125] Preferably, the term "reference value" as used herein refers to a predetermined value. The predetermined value should allow for the assessments referred to herein, such as in the diagnosis of myocardial tissue damage, the determination of the extent of myocardial tissue damage, the diagnosis of atrial arrhythmias, and / or the assessment of mitral regurgitation.

[0126] For example, in methods for diagnosing myocardial tissue injury, the reference, i.e., the reference value, should allow for differentiation between subjects with and without such injury. For example, in methods for diagnosing atrial fibrillation (AF), the reference should allow for differentiation between subjects with and without AF. In methods for assessing mitral regurgitation, the reference should allow for differentiation between the absence of mitral regurgitation and mitral regurgitation, or between any of the following: absence, grade I, grade II, or grade III mitral regurgitation.

[0127] Based on the diagnosis of myocardial tissue damage, the preferred diagnostic algorithm is as follows: Preferably, the amount of biomarker MyL7 and optionally at least one other biomarker is higher than that of a subject with a reference indication of myocardial tissue damage, and / or the amount of biomarker MyL7 and optionally at least one other biomarker is lower than that of a subject without myocardial tissue damage.

[0128] Based on the diagnosis of atrial arrhythmias, the optimal diagnostic algorithm is as follows: Preferably, the amount of biomarker MyL7 and optionally at least one other biomarker is higher than that of a subject with a reference indication of atrial arrhythmia, and / or the amount of biomarker MyL7 and optionally at least one other biomarker is lower than that of a subject without atrial arrhythmia.

[0129] Based on the assessment of mitral regurgitation, the optimal diagnostic algorithm is as follows: The reference level can also be the amount of biomarker in a sample obtained from a patient known to have mitral regurgitation or a predetermined degree of mitral regurgitation (grade I to III). A biomarker level in a test sample that is the same as or higher than the reference level should indicate the presence of mitral regurgitation or some degree thereof.

[0130] Furthermore, the reference value may depend on the time point at which the sample was collected (e.g., after surgery). Technicians will take this into account.

[0131] The comparison step may include calculating the ratio of a determined amount of the biomarker. For example, the ratio of the amount in the test sample to a reference amount (e.g., the amount in a first sample) is calculated. Based on this ratio, myocardial tissue damage is assessed. For example, when the ratio is in the range of 1.5 to 200,000, the amount of the biomarker in the patient is defined as an increase compared to the reference amount. In various embodiments, "increased amount" means an increase of at least 1.5 times relative to the reference amount, preferably at least 2 times. Preferably, "increased amount" means an increase of at least 100 times relative to the reference amount, particularly at least 1000 times. In embodiments, the increase is at least 10,000 times, or even at least 50,000 times, relative to the reference amount.

[0132] The present invention further relates to a method for monitoring cardiac surgery, the method comprising: a) Determine the amount of the biomarker MyL7 in the first sample obtained from the subject prior to cardiac surgery. b) Determine the amount of the biomarker MyL7 in a second sample obtained from the subject within 12 hours after the cardiac surgery. c) Compare the amount in the second sample with the amount in the first sample.

[0133] In an embodiment of the method of the present invention, the method further includes the step of having the patient undergo the cardiac surgery.

[0134] The present invention also relates to the use of a biomarker MyL7 (myosin light chain 7) or at least one detection agent for assessing mitral regurgitation, the detection agent specifically binding to said biomarker in at least one sample from a subject.

[0135] Furthermore, the present invention provides a method for assessing mitral regurgitation in subjects, the method comprising: a) Determine the amount of MyL7 in samples from subjects suspected of having mitral regurgitation; b) Compare the determined MyL7 value with a reference; and c) Assess mitral regurgitation based on the aforementioned comparison.

[0136] Preferably, the assessment includes diagnosing whether the subject has mitral regurgitation. More preferably, the assessment includes differentiating between no mitral regurgitation, grade I mitral regurgitation, grade II mitral regurgitation, and grade III mitral regurgitation.

[0137] Furthermore, this invention relates to the in vitro use of the biomarker MyL7 (myosin regulatory light chain 2, atrial subtype) or at least one detection agent for monitoring cardiac surgery, the detection agent specifically binding to the biomarker described in the following categories. a) Samples obtained from the subject before surgery, and b) Samples obtained from the subject within 12 hours after surgery.

[0138] As used herein, the term "monitoring cardiac surgery" preferably refers to assessing myocardial tissue damage (preferably atrial myocardial tissue damage) caused by said surgery. More preferably, the term refers to "monitoring the success of cardiac surgery," particularly its effectiveness, such as the technical success of the cardiac surgery (especially ablation). Therefore, it is about assessing whether the surgery (especially ablation) has been successful. Subjects who have undergone successful cardiac surgery have a lower risk of recurrence of atrial fibrillation (compared to the average risk after cardiac surgery). Subjects who have undergone unsuccessful cardiac surgery have a higher risk of recurrence of atrial fibrillation (compared to the average risk after cardiac surgery). Close monitoring is preferably performed on such subjects. Furthermore, ablation procedures can be repeated.

[0139] The terms "subject" and "cardiac surgery" have been defined above. These definitions apply accordingly. In a preferred embodiment of the method described above, 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.

[0140] Endocardial ablation or epicardial ablation is well known in the art and is described, for example, in Parameswaran et al. (Nat Rev Cardiol 18, 210-225 (2021)). An ablation line is established within the atria of the heart according to a specific protocol to prevent the conduction of electrical impulses that would cause atrial fibrillation instead of regular contraction. This ablation can be performed as an endocardial ablation following incision of the right and left atria, or as a concomitant epicardial ablation of the pulmonary veins. Typically, endocardial ablation is endocardial cryoablation. Typically, epicardial ablation is radiofrequency ablation. Endocardial ablation is a more invasive form of ablation than epicardial ablation and results in greater myocardial damage (see EP 3 775 914 A1).

[0141] The first sample serves as a reference sample. It should be obtained before the surgery. Preferably, the first sample is obtained within one week before the surgery, more preferably within three days before the surgery, and most preferably within one day before the surgery.

[0142] The second sample should be obtained within 12 hours after surgery, more preferably within 8 hours after surgery, even more preferably within 4 hours after surgery, and most preferably within 3 hours after surgery.

[0143] In another preferred embodiment, the second sample is obtained at least one hour but no later than eight hours after the cardiac surgery. In yet another preferred embodiment, the sample is obtained at least one hour but no later than four hours after the cardiac surgery. In still another preferred embodiment, the sample is obtained at least one hour but no later than three hours after the cardiac surgery.

[0144] Furthermore, it is envisioned that the second sample is obtained at a predetermined time after the cardiac surgery. For example, the sample may be obtained approximately 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 the surgery.

[0145] In a preferred embodiment, the second sample is obtained approximately 2 hours after the surgery.

[0146] In this embodiment, the surgery is completed when the subject is admitted to the ICU (Intensive Care Unit), that is, at the point when the patient arrives at the ICU.

[0147] Compared with the amount of biomarkers in the first sample, the amount of biomarkers in the second sample is preferably increased, more preferably significantly increased, and most preferably statistically significantly increased, indicating atrial myocardial tissue damage caused by surgery.

[0148] The amount of biomarkers in the second sample is preferably increased compared to the amount in the first sample, and more preferably significantly increased, and most preferably statistically significantly increased, indicating that the cardiac surgery has been successful.

[0149] Preferably, "increase" as a significant increase means an increase of at least 100 times relative to a reference amount, and more particularly, an increase of at least 1000 times. In embodiments, the increase is at least 10,000 times relative to a reference amount, or even at least 50,000 times.

[0150] If the increase is less than the indications above, such as less than 100 times the increase, the cardiac surgery is unsuccessful. In this case, the subject should be closely monitored. Alternatively, the subject may undergo ablation surgery again.

[0151] The present invention further relates to a method for determining the amount of the biomarker MyL7 and, optionally, the amount of at least one other biomarker, said other biomarker being cardiac troponin and / or cMyBPC, said method comprising: a) Provide at least one sample from the subject, wherein the sample was obtained from the subject within 12 hours after cardiac surgery, and b) In at least one sample provided in step a), determine the amount of biomarker MyL7 and optionally, the amount of at least one additional biomarker, said additional biomarker being cardiac troponin and / or cMyBPC.

[0152] The present invention further relates to the use of the biomarker MyL7 (myosin regulatory light chain 2, atrial subtype) or at least one detection agent for assessing myocardial tissue damage (preferably, in vitro), the detection agent specifically binding to said biomarker in at least one sample from a subject. In a preferred embodiment of this use, the use further includes the use of: i) cardiac troponin or a detection agent specifically binding to cardiac troponin, and / or ii) cMyBPC or a detection agent specifically binding to cMyBPC.

[0153] The present invention further relates to the use (preferably, in vitro) of at least one detection agent that specifically binds to MyL7 for determining the amount of MyL7 in a sample obtained from a subject within 12 hours after cardiac surgery. In a preferred embodiment of this use, the use further includes the use of: i) cardiac troponin or a detection agent that specifically binds to cardiac troponin to determine the amount of cardiac troponin, and / or ii) cMyBPC or a detection agent that specifically binds to cMyBPC to determine the amount of cMyBPC.

[0154] The present invention further relates to a kit for assessing myocardial tissue damage, the kit comprising at least one assay that specifically binds to MyL7, and optionally at least one assay that specifically binds to cardiac troponin and / or at least one assay that specifically binds to cMyBPC. Typically, the kit is formulated to suit the implementation of the method of the present invention. Typically, the kit further includes instructions for use to implement the method.

[0155] As used herein, the term "kit" refers to a collection of the aforementioned components, preferably provided separately or in a single container. The container also includes instructions for carrying out the methods of the invention. These instructions may be in the form of a manual or may be provided by computer program code capable of implementing the comparisons mentioned in the methods of the invention and establishing assessments accordingly when implemented on a computer or data processing device. Furthermore, the kit should include at least one standard as defined above, i.e., a solution having a predefined amount of one or more biomarkers mentioned herein, the predefined amount representing a reference amount.

[0156] In some embodiments, the kits disclosed herein include at least one component or a package of components for performing the disclosed methods. “Package combination” means that these kits provide a single package containing a combination of one or more components, such as probes (e.g., antibodies or antigen-binding fragments thereof) disclosed herein, controls, buffers, reagents (e.g., conjugates and / or substrates), instructions, etc. The definition of “packaging combination” also includes kits containing a single container. In some embodiments, these kits include at least one probe, such as an antibody (having specific affinity for the epitopes of the biomarkers disclosed herein). For example, these kits may include fluorophore-labeled antibodies or antibodies that are members of a fusion protein. In the kit, the probe may be immobilized and may be immobilized in a specific conformation. For example, immobilized probes may be provided in the kit for specific binding to a target protein, for detecting the target protein in a sample, and / or for removing the target protein from a sample.

[0157] The containers for these kits can be any container suitable for packaging and / or containing one or more components disclosed herein, including, for example, probes (e.g., antibodies), controls, buffers, and reagents (e.g., conjugates and / or substrates). Suitable materials include, but are not limited to, glass, plastic, cardboard or other paper products, wood, metals, and any alloys thereof. In some embodiments, the container may completely enclose one or more immobilized probes, or may simply cover the probes to minimize contamination from dust, grease, etc., and light exposure. In some further embodiments, these 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 from other containers, or different from some but not all other containers.

[0158] It should be understood that, as used in the specification and claims, "a" or "an" may mean one / a type or a plurality / multiple types, depending on the context in which it is used. Thus, for example, a reference to an item "a" may mean that at least one item can be utilized.

[0159] As used herein, the terms “have,” “comprise,” or “include,” or any grammatical variations thereof, are used in a non-exclusive manner. Thus, these terms can refer either to a situation where no further features exist in the entity described in this context besides the features introduced by these terms, or to a situation where one or more further features exist. For example, the statements “A has B,” “A contains B,” and “A includes B” can refer to a situation where no other elements exist in A besides B (i.e., where A is solely and uniquely composed of B), or to a situation where one or more further elements (such as elements C, C and D, or even further elements) exist in entity A besides B. The term “comprise” also covers instances where only the mentioned items exist, i.e., it has a restrictive meaning in the sense of “composed of.”

[0160] Furthermore, as used herein, the terms “particularly,” “more particularly,” “typically,” and “more typically,” or similar terms, are used in conjunction with additional / alternative features without limiting the possibility of substitution. Therefore, features introduced by these terms are additional / alternative features and are not intended to limit the scope of the claims in any way. As those skilled in the art will recognize, the invention can be practiced using alternative features. Similarly, features introduced by “in embodiments of the invention” or similar expressions are intended to be additional / alternative features without limiting alternative embodiments of the invention, without limiting the scope of the invention, and without limiting the possibility of combining features introduced in this way with other additional / alternative or non-additional / alternative features of the invention.

[0161] Furthermore, it should be understood that the term "at least one" as used herein means that one or more of the items mentioned after the term may be used according to the invention. For example, if the term indicates that at least one sampling unit should be used, it can be understood as one sampling unit or more than one sampling unit, i.e., two, three, four, five, or any other number. Depending on the item referred to by the term, those skilled in the art will understand the upper limit (if any) that the term may refer to.

[0162] As used herein, the term "about" means, relative to any number referenced after the term, an interval of precision that enables the achievement of the technical effect. Therefore, "about" as used herein preferably refers to a precise value or a range of ±20%, preferably ±15%, more preferably ±10%, or even more preferably ±5% of the precise value.

[0163] All publicly available information in all references cited in this specification, as well as any publicly available information specifically mentioned herein, is incorporated herein by reference.

[0164] Embodiments of the present invention The following embodiments are preferred embodiments of the present invention. The definitions provided above are subject to necessary modifications.

[0165] 1. The use of a biomarker MyL7 (myosin light chain 7) or at least one assay for assessing myocardial tissue damage, wherein the assay specifically binds to the biomarker in at least one sample from a subject.

[0166] 2. The use as described in Example 1, wherein the sample is blood, serum or plasma or interstitial fluid.

[0167] 3. The use as described in Example 1 or 2, wherein the subject is a human subject.

[0168] 4. The use according to any one of Examples 1 to 3, wherein the detection agent is an antibody or an antigen-binding fragment thereof.

[0169] 5. The use according to any one of Examples 1 to 4, wherein the subject is suspected of having myocardial tissue damage.

[0170] 6. The use according to any one of Examples 1 to 5, wherein the myocardial tissue injury is atrial myocardial injury.

[0171] 7. The use according to any one of Examples 1 to 6, wherein the sample is obtained after cardiac surgery, particularly cardiac surgery involving the atria of the heart.

[0172] 8. According to the use described in Example 7, the cardiac surgery is cardiac ablation (such as endocardial ablation or epicardial ablation), left atrial appendage occlusion, or atrial septal shunt occlusion.

[0173] 9. The use according to Example 7 or 8, wherein the sample is obtained within 12 hours after surgery, such as within 8 hours, for example within 4 hours.

[0174] 10. The use according to any one of the foregoing embodiments, wherein the assessment of myocardial tissue damage is selected from... a) Diagnosis of myocardial tissue injury, b) Differentiation between atrial and ventricular myocardial tissue injury. c) Determination of the extent of myocardial tissue damage, and / or d) Diagnosis of atrial arrhythmias.

[0175] 11. The use according to any one of the foregoing embodiments further includes the use of: i) cardiac troponin or a detection agent that specifically binds to cardiac troponin, and / or ii) cMyBPC or a detection agent that specifically binds to cMyBPC.

[0176] 12. A method for assessing myocardial tissue damage, the method comprising determining the amount of a biomarker MyL7 in at least one sample from a subject, and assessing myocardial tissue damage based on the amount of said biomarker.

[0177] 13. The method according to Example 12, wherein the sample was obtained after cardiac surgery.

[0178] 14. The method according to Example 12 or 13, further comprising comparing the amount of biomarker with a reference amount.

[0179] 15. The method according to Example 14, wherein the reference quantity is a predetermined value.

[0180] 16. The method according to Examples 13 and 14, wherein the reference amount is the amount of biomarker in a sample obtained from the subject prior to cardiac surgery.

[0181] 17. The method according to any one of the foregoing embodiments further includes determining the amount of cardiac troponin and / or the amount of cMyBPC.

[0182] 18. A method for assessing myocardial tissue injury, the method comprising the following steps: a) Provide at least one sample from the subject, b) In the at least one sample provided in step a), determine the amount of biomarker MyL7 and, optionally, the amount of at least one additional biomarker, said additional biomarker being cardiac troponin and / or cMyBPC, and c) Provide the physician with information about the amount of the identified biomarker MyL7 and, optionally, the amount of the identified at least one other biomarker, thereby aiding in the assessment of myocardial tissue damage.

[0183] 19. A method for assessing myocardial tissue injury, the method comprising: a) Provides an assay for the biomarker MyL7 and, optionally, at least one additional assay for another biomarker, said other biomarker being cardiac troponin and / or cMyBPC, and b) Provide instructions on the use of the results obtained or available through the stated assay in this assessment of myocardial tissue damage.

[0184] 20. A computer-implemented method for assessing myocardial tissue injury, the method comprising: a) Receive at the processing unit a value for the amount of MyL7 and, optionally, at least one additional value for the amount of at least one other biomarker, said additional biomarker being cardiac troponin and / or cMyBPC, wherein said amount of MyL7 and, optionally, the amount of said at least one other biomarker, have been determined in at least one sample from the subject. b) The processing unit compares one or more values ​​received in step (a) with one or more reference values, and c) Assess myocardial tissue damage based on the comparison step b).

[0185] 21. The method according to any one of the foregoing embodiments further includes recommending or initiating a suitable therapy.

[0186] 22. A method for monitoring cardiac surgery, the method comprising: a) Determine the amount of the biomarker MyL7 in the first sample obtained from the subject prior to cardiac surgery. b) Determine the amount of the biomarker MyL7 in a second sample obtained from the subject within 12 hours after the cardiac surgery. c) Compare the amount in the second sample with the amount in the first sample.

[0187] 23. A method for diagnosing atrial fibrillation, the method comprising: a) Determine the amount of the biomarker MyL7 in at least one sample from the subject. b) Compare the quantity determined therefrom with a suitable reference quantity, and c) Diagnose atrial fibrillation in the subject based on the results of the comparison step.

[0188] 24. The method according to Example 23, wherein the diagnosis includes differentiating between sinus arrhythmia, paroxysmal atrial fibrillation and persistent or permanent atrial fibrillation.

[0189] 25. The method according to Example 24, wherein the identification is performed between sinus arrhythmia or paroxysmal atrial fibrillation on the one hand and persistent or permanent atrial fibrillation on the other hand.

[0190] 26. The use of a biomarker MyL7 (myosin light chain 7) or at least one assay for assessing mitral regurgitation, wherein the assay specifically binds to the biomarker in at least one sample from a subject.

[0191] 27. A method for assessing mitral regurgitation in a subject, the method comprising: a) Determine the amount of MyL7 in samples from subjects suspected of having mitral regurgitation; b) Compare the determined MyL7 value with a reference; and c) Assess mitral regurgitation based on the aforementioned comparison.

[0192] 28. The use as described in Example 26 or the method as described in Example 27, wherein the assessment includes diagnosing whether the subject has mitral regurgitation.

[0193] 29. The use as described in Example 26 or the method as described in Example 27, wherein the assessment includes distinguishing between no mitral regurgitation, Grade I mitral regurgitation, Grade II mitral regurgitation and Grade III mitral regurgitation.

[0194] 30. A kit for assessing myocardial tissue damage, the kit comprising at least one detection agent that specifically binds to MyL7, and optionally at least one detection agent that specifically binds to cardiac troponin and / or at least one detection agent that specifically binds to cMyBPC.

[0195] 31. The use of at least one assay agent that specifically binds to MyL7 for determining the amount of MyL7 in a sample obtained from a subject within 12 hours after cardiac surgery.

[0196] 32. A method for determining the amount of biomarker MyL7 and optionally, the amount of at least one other biomarker, said other biomarker being cardiac troponin and / or cMyBPC, said method comprising: a) Provide at least one sample from the subject, wherein the sample was obtained from the subject within 12 hours after cardiac surgery, and b) In at least one sample provided in step a), determine the amount of biomarker MyL7 and optionally, the amount of at least one additional biomarker, said additional biomarker being cardiac troponin and / or cMyBPC.

[0197] Example The present invention is illustrated by the following examples only. In any case, these examples should not be interpreted in a way that limits the scope of the invention.

[0198] Example 1 A robust prototype assay (RPA) using monoclonal antibodies was developed for the biomarker MyL7 and evaluated in multiple cohorts (see Examples 2 through 4). Cardiac troponin T and cMyBPC were evaluated as reference biomarkers reflecting overall myocardial injury.

[0199] Example 2: Mapping Queue MyL7, cMyBPC, and TnThs levels were determined in plasma samples from n=60 patients who underwent open-heart surgery prior to CABG or valve surgery, and biomarker measurements were performed. Evidence of different types of atrial fibrillation (AF) (paroxysmal AF, n=14; persistent AF, n=16) or spontaneous atrial fibrillation (SR) (control, n=30) was generated during surgery using simultaneous endocardial-epidermal high-density activation mapping.

[0200] Based on the different conduction patterns of fibrillation waves induced by electrical activation, epicardial high-density mapping is another means of differentiating between sinus rhythm, paroxysmal, and persistent atrial fibrillation (see Eckstein et al., Transmural Conduction Is the Predominant Mechanism of Breakthrough During Atrial Fibrillation Evidence From Simultaneous Endo-Epicardial High-DensityActivation 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. JAFIB: Journal of Atrial Fibrillation; August / September 2015, Vol. 8, No. 2, p. 5).

[0201] These results are shown in Figure 1.

[0202] Data assessment showed that patients with MyL7 levels above the reference range were suspected of having atrial fibrillation (AF). The observed AUC for MyL7 in detecting paroxysmal or persistent AF was 0.73. In contrast, the observed AUC for cMyBPC in detecting paroxysmal or persistent AF was 0.55. Therefore, in the patients analyzed in this study, the biomarker MyL7 allows for improved detection of AF compared to cMyBPC.

[0203] Therefore, in the patients analyzed in this study, MyL7 performed significantly better than cMyBPC. Assessment of MyL7 in the MAPPING study showed that moderately elevated circulating MyL7 titers were associated with persistent atrial pulmonary angina (AF), a condition associated with mild atrial muscle tissue damage.

[0204] Evaluation of MyL7 in the MAPPING study showed that circulating atrial myoprotein MyL7 or fragments thereof were detected in diseases associated with spontaneous atrial muscle injury.

[0205] Example 3: MAZE queue MyL7, cMyBPC, and TnThs levels were determined in plasma samples from n=52 enrolled patients who underwent open-chest surgery. These patients received continuous blood sampling at baseline and at 2h, 4h, 6h, and 24h postoperatively. Patients underwent open-chest surgery with either epicardial or endocardial ablation (see Lahm et al. 2019, ibid.). Following induced atrial injury, MyL7 was released into the bloodstream, reaching peak levels approximately two hours postoperatively. The increase in circulating MyL7 levels correlated with the degree of atrial injury (endocardial or epicardial).

[0206] These results are shown in Figure 2.

[0207] Data assessment in patients who underwent endocardial ablation showed a significant increase in circulating levels of all biomarkers after intervention. However, the data clearly demonstrated that the relative change in MyL7 was much higher compared to cMyBPC and TnThs.

[0208] Authors Lahm et al. (2019) measured MyBPHL in samples from the MAZE cohort, another cardiac biomarker presumed to be atrial and ventricular specific. Published data in patients undergoing endocardial ablation showed that MyBPHL values ​​observed at ICU admission were approximately 3 to 4 times higher than preoperative MyBPHL values.

[0209] The MYL7 value observed upon ICU admission was approximately 180,000 times higher than the preoperative MyBPHL value. It is suggested that the TnThs / Myl7 ratio or Myl7 alone can be used to assess the severity of surgery-related atrial muscle tissue damage.

[0210] Assessment of MyL7 in the MAZE study showed that circulating atrial myoprotein MyL7 or fragments thereof were detected, and the severity of surgery-induced atrial muscle tissue damage was assessed.

[0211] Example 4: RRTAK queue MyL7, cMyBPC, and TnThs levels were determined in plasma samples from n=51 enrolled patients who underwent percutaneous aortic valve replacement surgery. These patients received consecutive blood samples at baseline and at 4h, 8h, 24h, 48h, 72h, 1 week, and 3 months postoperatively.

[0212] Following percutaneous aortic valve replacement, cardiac muscle proteins showed only a moderate increase in circulation. After intervention, serum MyL7 levels decreased more rapidly than TnThs. The TnThs / MyL7 ratio can be used to assess the severity of surgery-related atrial muscle tissue damage.

[0213] The assessment of MyL7 in the RRTAK study aimed to demonstrate that no significant increase in MyL7 titers was observed in patients receiving nonspecific cardiac interventions located in the atria.

[0214] Example 5: Elevated MYL7 is an indicator of mitral regurgitation. As described in Example 4, MYL7 levels in blood samples from patients undergoing RRTAK were analyzed. Mitral regurgitation is caused by regurgitation of blood into the left atrium, leading to increased volume and pressure. Patients undergoing the RRTAK study presented with varying degrees of mitral regurgitation: no regurgitation, grade I, II, and III.

[0215] The levels of MYL7 found in the blood samples of study participants were correlated with the presence and severity of mitral regurgitation. Low levels were found in patients without mitral regurgitation, while the highest levels were found in patients with grade II mitral regurgitation (Note: the data are not convincing given the small number of participants exhibiting grade III mitral regurgitation). The results are shown in Figure 3.

[0216] Example 6: Compared with paroxysmal atrial fibrillation or sinus arrhythmia, patients with persistent atrial fibrillation had elevated MYL7 levels. Atrial fibrillation is associated with fibrotic remodeling of the left atrium. In a study (Mapping), MYL7 levels were determined in patients with sinus arrhythmia, paroxysmal atrial fibrillation, and persistent atrial fibrillation.

[0217] High levels of MYL7 were found in patients with persistent atrial fibrillation, while significantly lower levels were found in patients with sinus arrhythmia or paroxysmal atrial fibrillation. The results are shown in Figure 4.

Claims

1. Use of a biomarker MyL7 (myosin light chain 7) or at least one assay for assessing myocardial tissue damage, wherein the assay specifically binds to the biomarker in at least one sample from a subject.

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

3. The use according to claim 1 or 2, wherein the subject is a human subject, and / or wherein the subject is suspected of having myocardial tissue damage.

4. The use according to any one of claims 1 to 3, wherein the myocardial tissue injury is atrial myocardial injury.

5. The use according to any one of claims 1 to 4, wherein the sample is obtained after cardiac surgery, particularly cardiac surgery involving the atria of the heart, such as said cardiac ablation (e.g., endocardial ablation or epicardial ablation), left atrial appendage occlusion, or atrial septal shunt occlusion.

6. The use according to claim 5, wherein the sample is obtained within 12 hours, such as within 8 hours, for example within 4 hours, after the cardiac surgery.

7. The use according to any one of the preceding claims, wherein the assessment of myocardial tissue injury is selected from... a) Diagnosis of myocardial tissue injury, b) Differentiation between atrial and ventricular myocardial tissue injury. c) Determination of the extent of myocardial tissue damage, and / or d) Diagnosis of atrial arrhythmias.

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

9. The method of claim 8, wherein a) The sample was obtained after cardiac surgery. b) The method further includes comparing the amount of the biomarker with a reference amount, particularly wherein the reference amount is a predetermined value, or wherein the reference amount is the amount of the biomarker in a sample obtained from the subject prior to cardiac surgery.

10. A method for assessing myocardial tissue injury, the method comprising the following steps: a) Provide at least one sample from the subject, b) In the at least one sample provided in step a), determine the amount of biomarker MyL7 and, optionally, the amount of at least one additional biomarker, said additional biomarker being cardiac troponin and / or cMyBPC, and c) Provide the physician with information about the amount of the identified biomarker MyL7 and, optionally, the amount of the identified at least one other biomarker, thereby aiding in the assessment of myocardial tissue damage.

11. A method for assessing myocardial tissue injury, the method comprising: a) Provides an assay for the biomarker MyL7 and, optionally, at least one additional assay for another biomarker, said other biomarker being cardiac troponin and / or cMyBPC, and b) Provide a description of the use of the results obtained or available through the assay in the assessment of myocardial tissue damage.

12. A computer-implemented method for assessing myocardial tissue damage, the method comprising: a) Receive at the processing unit a value for the amount of MyL7 and, optionally, at least one additional value for the amount of at least one other biomarker, said additional biomarker being cardiac troponin and / or cMyBPC, wherein said amount of MyL7 and, optionally, said amount of said at least one other biomarker, have been determined in at least one sample from the subject. b) The processing unit compares one or more values ​​received in step (a) with one or more reference values, and c) Based on the comparison step b), assess myocardial tissue damage, the assessment including recommending or initiating appropriate treatment.

13. A method for monitoring cardiac surgery, such as cardiac ablation, the method comprising: a) Determine the amount of the biomarker MyL7 in the first sample obtained from the subject prior to cardiac surgery. b) Determine the amount of the biomarker MyL7 in a second sample obtained from the subject within 12 hours after the cardiac surgery. c) Compare the amount in the second sample with the amount in the first sample.

14. A method for diagnosing atrial fibrillation, the method comprising: a) Determine the amount of the biomarker MyL7 in at least one sample from the subject. b) Compare the quantity thus determined with a suitable reference quantity, and optionally... c) Diagnose atrial fibrillation in the subject based on the results of the comparison step.

15. The method of claim 14, wherein the diagnosis includes differentiating between sinus arrhythmia, paroxysmal atrial fibrillation, and persistent or permanent atrial fibrillation.

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

17. The use of a biomarker MyL7 (myosin light chain 7) or at least one assay for assessing mitral regurgitation, wherein the assay specifically binds to the biomarker in at least one sample from a subject.

18. A method for assessing mitral regurgitation in a subject, the method comprising: a) Determine the amount of MyL7 in samples from subjects suspected of having mitral regurgitation; b) Compare the determined MyL7 value with the reference value; as well as c) Assess mitral regurgitation based on the aforementioned comparison.

19. The use according to claim 17 or the method according to claim 18, wherein the assessment includes diagnosing whether the subject has mitral regurgitation.

20. The use according to claim 17 or the method according to claim 18, wherein the assessment includes distinguishing between no mitral regurgitation, Grade I mitral regurgitation, Grade II mitral regurgitation and Grade III mitral regurgitation.

21. A kit for assessing myocardial tissue damage, the kit comprising at least one detection agent that specifically binds to MyL7, and optionally at least one detection agent that specifically binds to cardiac troponin and / or at least one detection agent that specifically binds to cMyBPC.

22. The use of at least one detection agent that specifically binds to MyL7 for determining the amount of MyL7 in a sample obtained from a subject within 12 hours after cardiac surgery, such as after cardiac ablation.

Images