CROSS-REFERENCE TO RELATED APPLICATIONS
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This application is a continuation of U.S. patent application Ser. No. 12/440,226, filed Aug. 25, 2009, now U.S. Pat. No. 8,153,376, issued Apr. 10, 2012, which is a national phase entry of PCT/EP2007/060173, filed Sep. 25, 2007, and published in English as International Patent Publication WO 2008/037720 A2 on Apr. 3, 2008, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 06121196.7, filed Sep. 25, 2006, and European Patent Application Serial No. 06121525.7, filed Sep. 29, 2006, the disclosure of each of the above-referenced priority documents is hereby incorporated herein by this reference in its entirety.
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The present invention, in general, relates to the field of medicine, more specifically, the field of cardiology. The invention, in particular, relates to means and methods for diagnosing and/or treating subjects at risk of developing heart failure.
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It is generally known that chronic cardiac loading, as occurs during long-standing hypertension, valvular disease or other chronic disorders like diabetes, induces cardiac hypertrophy, which is one of the most important risk factors for heart failure. Congestive heart failure (HF) is a common but severe and complex clinical syndrome, especially among elderly people, characterized by a diminished cardiac contractile function and decreased exercise tolerance. Symptoms of heart failure include, amongst others, pulmonary and peripheral edema, fatigue and/or dyspnea. Severe heart failure may also lead to reduced function in other organs because these are not receiving enough blood.
Not all hypertrophied hearts, however, will ultimately fail. Thus, while an important number of patients progress to developing life-threatening complications, others may remain stable for prolonged periods. Until now, the molecular changes that precede and herald this transition from hypertrophy towards heart failure are incompletely understood.
As early identification of patients at risk for developing hypertensive end organ damage, such as heart failure, may prevent rapid progression, it would be preferable to be able to identify (diagnose) those patients in which heart failure is likely to occur before it actually does so. Early diagnosed patients may thus be treated in order to prevent the onset of heart failure. In addition, it would be preferable to be able to identify those patients suffering from heart failure who are at risk for developing severe complications.
Current methods can reliably exclude the actual presence of heart failure, but cannot reliably prove the existence of heart failure, nor can these methods predict the outcome of established heart failure, or predict the occurrence of heart failure.
A need, therefore, exists for simple and reliable methods for predicting the likelihood of onset of heart failure and/or for predicting the outcome of already established heart failure. In addition, the development of means and methods for treating patients who are at risk of developing heart failure, before heart failure and/or its complications occur, would be of great clinical importance.
The object of the present invention is to provide diagnostic methods by which patients can be identified who are at particular risk of developing heart failure and/or who are at particular risk to develop complications of heart failure. It is a further object of the present invention to provide means and methods for treating patients who are at risk of developing heart failure and/or who are at risk for developing complications of heart failure.
This objective is achieved by the present invention by providing a method for diagnosing a subject at risk of developing heart failure, comprising the steps of:
(a) determining the level of one or more biological markers in a biological sample of the subject;
(b) comparing the level of the biological marker(s) to a standard level of the same biological marker(s); and
(c) determining whether the level of the biological marker(s) is indicative of a risk for developing heart failure,
wherein the biological marker is lysosomal integral membrane protein-2 (LIMP-2) and/or Krüppel-Like Transcription Factor 15 (KLF15).
In the research that led to the present invention, a number of genes have been identified that are involved in the development of heart failure. The identified genes have been listed in Table 2. It has furthermore been demonstrated that specific polypeptides encoded by the genes are indeed mechanistically linked to heart failure. It has, in particular, been demonstrated that specific proteins encoded by the genes from Table 2 are involved in the molecular mechanisms that are responsible for the transition from cardiac hypertrophy towards heart failure, and thus can be used as a biological marker for identifying patients at risk of developing heart failure. In addition, these proteins, and/or the genes encoding these proteins, and/or polypeptide and/or polynucleotide fragments or variants of these proteins and/or genes, can be used as a target for treating those patients at risk.
According to the present invention, it has, in particular, been demonstrated that specific intercalated disc components, in particular, lysosomal integral membrane protein-2 (LIMP-2) and Krüppel-Like Transcription Factor 15 (KLF15) are involved in the molecular mechanisms that predict the transition from cardiac hypertrophy towards heart failure, and can suitably be used as biological markers (biomarkers) for the identification of individuals who are at risk of developing heart failure.
According to the present invention, it has thus been found that subjects at risk for developing heart failure can be identified by determining the level of one or more of the identified biological markers in a biological sample of the subject and comparing the level of the marker to a standard level. The standard level is derived from healthy subjects, i.e., the standard level is the level of the biological marker in the biological sample of healthy persons, i.e., persons free from cardiac disease. If the level of the biological marker tested is altered, e.g., elevated or reduced (depending on the specific biological marker concerned) compared to the standard level, the subject is at risk for developing HF and/or for developing severe complications of heart failure.
An early diagnosis of heart failure, preferably before clinical symptoms occur, is essential for, e.g., successfully addressing underlying diseases and/or preventing further myocardial dysfunction and clinical deterioration by, for example, treatment of the diagnosed patients.
In the research that led to the present invention, the gene expression profile of a large number of genes from hearts that were hypertrophied due to high blood pressure, but appeared functionally well and compensated by traditional techniques (echocardiography) but later proved to develop heart failure, were investigated. This expression profile was compared to the gene expression profile obtained from hearts that that were also hypertrophied due to high blood pressure and appeared equally functionally well and compensated by traditional techniques (echocardiography), but later proved NOT to develop heart failure and remained stable. This way, genes were identified that predicted the occurrence of later developing heart failure, which, according to the present invention, have been shown to be novel and crucial modulators of hypertrophy and the transition toward heart failure. These genes have been listed in Table 2. Subsequently, specific preferred biological markers, in particular, specific intercalated disc-related biological markers were identified. The intercalated disc (ID) forms the connection between cardiac myocytes making up the cardiac fibers in the heart. The intercalated disc thus is a specialized cell-cell junction providing mechanical and electrical coupling between the cells and supporting synchronized contraction of cardiac tissue.
According to the present invention, it has thus been demonstrated that increased cardiac expression of LIMP-2, as compared to standard levels of expression, identifies those hypertrophied hearts that are prone to progress to overt heart failure. Thus, while cardiac development is normal in LIMP-2 null mice (Gamp et al., 2003), hypertension induced dilated cardiomyopathy in these mice. It was shown that LIMP-2 binds to the vital cardiac adherens junction protein N-cadherin and is essential to secure proper interactions between N-cadherin and β-catenin. It has further been found that expression of LIMP-2 is increased in hypertrophied rat hearts that are on the brink of progressing to heart failure, thus suggesting that increased LIMP-2 expression by cardiac myocytes heralds their inability to normalize mechanical forces. As such, increased LIMP-2 expression may be seen as a desperate attempt of the myocyte to respond to worsening loading and be indicative of imminent failure. It has moreover been shown that LIMP-2 expression is significantly increased in patients with clinically severe pressure loading. By determining the level of LIMP-2 protein and/or the level of expression of the gene coding for LIMP-2 in hypertensive subjects, and comparing these level(s) with a standard level, and subsequently determining whether the level is indicative of a risk for developing heart failure, it thus is possible to identify in a very early stage the myocardium that is about to succumb to the pressure. In particular, an increased level of LIMP-2 protein and/or an increased level of LIMP-2 gene expression as compared to a standard level is indicative of a risk for developing heart failure and/or heart failure-related complications.
In the research that led to the present invention, it has further been shown that the gene coding for Krüppel-Like Factor 15 (KLF15) characterized hypertrophied hearts that quickly progressed to heart failure. This was confirmed by real-time PCR, which showed that KLF15 was down-regulated in compensated LVH, but that KLF-15 was significantly further suppressed in the hypertrophied hearts that quickly progressed to failure. It was further shown that KLF15 has a role in cardiac myocytes as a suppressor of cardiac hypertrophy. Determining the level of the KLF15 protein and/or the level of expression of the gene coding for KLF15 in hypertensive subjects, and comparing these levels to standard levels, thus also is useful for identifying in a very early stage those patients that are likely to develop heart failure. In the case of KLF15, a decreased level of KLF15 protein and/or decreased KLF15 gene expression in a biological sample, as compared to standard levels, is indicative for the development of heart failure.
The present invention relates both to in vivo methods, i.e., methods wherein the level of the biological marker is determined in a biological sample in vivo and to in vitro methods.
In a preferred embodiment of the invention, the level of the biological marker is determined in vitro in a biological sample obtained from an individual. For in vitro determining the level of the biological markers of the present invention, any suitable biological sample of any bodily fluid that may comprise a biological marker identified by this research may be used. Preferably, the biological sample is selected from the group consisting of blood, plasma, serum, or cardiac tissue. More preferably, the biological sample is a peripheral blood sample, or a plasma or serum sample derived from peripheral blood. Peripheral blood samples can, e.g., easily be taken from the patients and do not need complex invasive procedures such as catheterization. The biological sample may be processed according to well-known techniques to prepare the sample for testing.
For measuring the level of the biological markers of the invention, use can be made of conventional methods known in the art.
When the biological marker is a protein and/or a fragment and/or a variant thereof, several conventional methods for determining the level of a specific protein, and/or fragments and/or variants thereof, which are well-known to the skilled person, may be used. The level of the marker may, for example, be measured by using immunological assays, such as enzyme-linked immunosorbent assays (ELISA), thus providing a simple, reproducible and reliable method. Antibodies for use in such assays are available, and additional (polyclonal and monoclonal) antibodies may be developed using well-known standard techniques for developing antibodies. Other methods for measuring the level of the biological protein markers may furthermore include (immuno)histochemistry, Western blotting, flow-cytometry, RIA, competition assays, etc., and any combinations thereof. In vivo, the level of, for example, non-secreted proteins can be determined by labeling and tagging specific antibodies against one of the proteins of interest. This allows visualization of the amount of protein in the heart by so called “molecular imaging” techniques.
When the biological marker is a gene, and/or a polynucleotide fragment and/or variant thereof, e.g., DNA, cDNA, RNA, mRNA, etc., such as a gene coding for a specific protein, or mRNA that is transcribed, the biological marker can be measured in, e.g., cardiac biopsies, by, e.g., well-known molecular biological assays, such as in situ hybridization techniques using probes directed to the specific polynucleotides. Other nucleic acid-based assays that may be used according to the invention include RT-PCR, nucleic acid-based ELISA, Northern blotting, etc, and any combinations thereof.