CROSS-REFERENCE TO RELATED APPLICATIONS
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.
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.
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.
In order to enhance the specificity and/or sensitivity of the diagnostic method, the method of the invention may include the detection of the level of one or more other (biological) markers, i.e., the detection of the biological markers of the present invention may suitably be combined with the detection of other markers that are indicative for the development of heart failure.
The present invention further relates to kits for performing the diagnostic methods as described above. The invention, in particular, relates to such diagnostic kits for identifying a subject at risk of developing heart failure, comprising means for receiving one or more biological samples of the subject, and means for determining the level of the biological marker(s) in the biological sample of the subject. Thus, a kit is provided that can be used as a reliable and easy diagnostic tool. The means for receiving the biological sample may, for example, comprise a well of a standard microtiter plate. The means for determining the level of an intercalated disc-related biological marker in the biological sample of the subject may, for example, comprise one or more specific antibodies, polynucleotide probes, primers, etc., suitable for detecting the biological marker(s), identified according to the present invention. The kits may further also comprise calibration means and instruction for use.
The invention also relates to the use of the biological markers of the present invention and/or fragments and/or functional variants thereof in a screening method for identifying compounds for the prevention and/or treatment of heart failure. In a particular embodiment, the method for identifying a compound for prevention and/or treatment of heart failure comprises:
(a) contacting one or more compounds with a polypeptide encoded by a polynucleotide listed in Table 2, preferably KLF15 and/or LIMP-2, and/or fragments, and/or variants thereof;
(b) determining the binding affinity of the compound to the polypeptide;
(c) contacting a population of mammalian cells with the compound that exhibits a binding affinity of at least 10 micromolar; and
(d) identifying the compound that is capable of prevention and/or treatment of heart failure.
The polypeptides to be tested in the screening method of the present invention may be tested in vitro, e.g., free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly, or in vivo.
To perform the methods, it is feasible to immobilize either the polypeptide of the present invention or the compound to facilitate separation of complexes from uncomplexed forms of the polypeptide, as well as to accommodate automation of the assay. Interaction (e.g., binding) of the polypeptide of the present invention with a compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
The binding affinity of the compound with the polypeptide can, e.g., be measured by methods known in the art, such as using surface plasma resonance biosensors (Biacore), by saturation binding analysis with a labeled compound (e.g., Scatchard and Lindmo analysis), via displacement reactions, by differential UV spectrophotometer, fluorescence polarization assay, Fluorometric Imaging Plate Reader (FLIPR®) system, Fluorescence resonance energy transfer, and Bioluminescence resonance energy transfer. The binding affinity of compounds can also be expressed in a dissociation constant (Kd) or as IC50 or EC50. The IC50 represents the concentration of a compound that is required for 50% inhibition of binding of another ligand to the polypeptide. The EC50 represents the concentration required for obtaining 50% of the maximum effect in vitro. The dissociation constant, Kd, is a measure of how well a ligand binds to the polypeptide; it is equivalent to the ligand concentration required to saturate exactly half of the binding sites on the polypeptide. Compounds with a high affinity binding have low Kd, IC50 and EC50 values, i.e., in the range of 100 nM to 1 pM; a moderate to low affinity binding relates to a high Kd, IC50 and EC50 values, i.e., in the micromolar range.
The present invention also relates to the use of the genes and/or proteins listed in Table 2, preferably of the KLF15 and/or LIMP-2 gene and/or protein, for the preparation of a medicament for a prophylactic and/or therapeutic medicament for the prevention and/or treatment of heart failure.
Preferably, the present invention relates to the use of a modulator of the genes and/or proteins listed in Table 2, preferably of the KLF15 and/or LIMP-2 gene and/or protein, for the preparation of a prophylactic and/or therapeutic medicament for the prevention and/or treatment of heart failure.
In the present application, a modulator may be any compound that stimulates the expression of and/or increases the level of one or more of the biological markers that are found to be reduced according to the invention (e.g., an agonist), or any compound that suppresses the expression and/or reduces the level of one or more of the biological markers that are found to be increased according to the invention (e.g., an antagonist).
The medicament may be a protein-based molecule, such as, for example, an antibody directed against the protein marker, and/or fragments and/or variants thereof. The present invention also includes chimeric, single chain and humanized antibodies, as well as Fab fragments and the products of a Fab expression library and Fv fragments and the products of an Fv expression library.
Alternatively, the medicament may be a nucleic acid-based molecule. The down-regulation of a gene can, for example, be achieved at the translational or transcriptional level using, e.g., antisense nucleic acids. Antisense nucleic acids are nucleic acids capable of specifically hybridizing with all or a part of a nucleic acid encoding a protein and/or the corresponding mRNA. The preparation of antisense nucleic acids, DNA encoding antisense RNAs, is known in the art. The medicament may also comprise small interfering (hairpin) RNA (siRNA). SiRNAs mediate the post-translational process of gene silencing by double-stranded DNA (dsDNA) that is homologous in sequence to the silenced RNA. The preparation of siRNAs is known in the art. Similarly, the up-regulation of a gene (or over-expression) may be achieved by several methods that are known in the art.
In a preferred embodiment of the present invention, the modulator is an inhibitor of TGFβ. According to the present invention, it has been shown that suppression of KLF-15 is a crucial step in the development of failure-prone forms of hypertrophy and that TGFβ strongly suppresses KLF-15. Inhibitors of TGFβ, which are currently being developed in different fields, thus may suitably be used for the development of a prophylactic and/or therapeutic medicament for the prevention and/or treatment of heart failure. Examples of suitable inhibitors of TGFβ that can be used according to the invention are TGFβ receptor inhibitors as made by Scios Inc., Los Angeles, Calif., U.S.A., who indicate on their website (world-wide web at sciosinc.com/scios/tgf): “Scios has developed novel and potent small molecule inhibitors designed to inhibit the action of TGF-beta at its receptor. These small molecules have been shown to be effective in reducing scar formation (fibrosis) when given orally to animals. Scios expects to advance two lead molecules, representing different chemical classes, into preclinical development that could potentially be used to treat disease conditions in patients with significant unmet medical needs.”
The present invention further relates to the use of the proteins identified according to the invention for generating diagnostic means for use in (molecular) imaging of one or more of the identified proteins to assess the level of the protein and thus identify a subject at risk of developing heart failure. The diagnostic means may, for example, comprise labeled antibodies directed against the biological protein markers.
The present invention is further illustrated by the following figures and Examples.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Increased expression of LIMP-2 in Ren-2 rats. FIG. 1A, A left ventricular cardiac biopsy was taken at age 10 weeks, when Ren-2 rats exhibit comparable cardiac hypertrophy and fractional shortening cannot distinguish rats that will progress to heart failure or stay compensated. Between 15 and 18 weeks of age, part of the Ren-2 rats developed heart failure and the remainder stayed compensated until sacrifice at 21 weeks of age. *, P<5e−6. FIG. 1B, LIMP-2 mRNA was found by microarray analysis in 10-week-old hypertrophic Ren-2 rats to be specifically over-expressed in failure-prone rats (HF-prone LVH, n=4), as compared to the hypertrophied LVs that remained compensated (comp LVH, n=6) and to controls (n=4). FIG. 1C, LIMP-2 protein was up-regulated in end-stage failing Ren-2 rats (HF, n=9), as compared to compensated Ren-2 rats (comp, n=6). Both failing and compensated Ren-2 rats had significantly elevated levels of LIMP-2 protein as compared to control rats (n=6). *, P<0.05 versus control; **, P<0.01 versus control; $, P<0.05 versus comp; Mwm, molecular weight marker; au, arbitrary units.
FIG. 2: AngII-treated LIMP-2 KO (KO Ang) mice have dilated cardiomyopathy. FIG. 2A, WT Ang mice (n=14) significantly increased their LV weight, while KO Ang (n=14) mice did not (*, P<0.01 versus WT (n=8) and KO Ang). In KO Ang mice, individual myocytes failed to increase their volume (WT and KO, n=4; WT Ang and KO Ang, n=5; myocyte area (au): 264±42, versus 308±14 in WT Ang; *, P<0.01). Bars represent 50 μm. FIG. 2B, LIMP-2 KO (n=3) and WT (n=4) mice showed comparable blood pressure responses to AngII. FIG. 2C, AngII-treated WTs (n=8) and KOs (n=8) showed comparable increases in BNP and ANF mRNA expression (*, P<0.05 versus baseline (n=4)), while aska mRNA expression was induced to a significantly lesser extent in KO Ang mice, consistent with their reduced myocyte volume (*, P<0.05 versus KO (n=4); $, P<0.05 versus WT Ang). FIG. 2D, Baseline echocardiographic parameters were similar for WT (n=10) and KO (n=11) mice (day 0). After 14 and 28 days of AngII, wild-type LV walls were significantly hypertrophied, while knockouts did not show hypertrophy and were even dilated (*, P<0.005 versus baseline and versus KO Ang; $, P<0.005 versus baseline and vs WT Ang). FIG. 2E, Beta-adrenergic response to dobutamin was decreased in KO Ang mice (WT and KO, n=4; WT Ang, n=14; KO Ang, n=9; *, P<0.005). LVW/BW, LV weight corrected for body weight.
FIG. 3: AngII-treated LIMP-2 KO mice have massive interstitial fibrosis. Sirius red staining of LVs of AngII-treated LIMP-2 knockout (n=4) and wild-type (n=5) mice shows marked interstitial fibrosis in knockout mice (*, P<0.02 versus WT Ang and KO baseline), while both knockout and wild-type mice treated with AngII show similar degree of perivascular fibrosis. Bars represent 250 μm.
FIG. 4: AngII-treated LIMP-2 KO mice show myocyte disarray. Desmin-stained cardiac myocytes of AngII-treated LIMP-2 KO mice show disarray and have a disturbed internal structure, as shown by the higher and more capricious desmin-expression in these mice. Bars represent 250 μm.
FIG. 5: LIMP-2 expression is up-regulated in other forms of cardiac stress. FIG. 5A, In neonatal rat cardiac myocytes, 6 hours stretch elevated LIMP-2 mRNA expression (n=4 per group). LIMP-2 mRNA was also up-regulated in hypertrophic myocardium (FIG. 5B) from rats that had undergone exercise training for 10 weeks (5 days per week, n=6) as compared to non-hypertrophic control rats (n=7) and from patients suffering from aortic stenosis (LVH, n=20) as compared to non-hypertrophic control patients (n=7). *, P<0.05 versus control; **, P<0.01 versus control; LVH; LV hypertrophy.
). Scale bar represents 1 μm. FIG. 6C, Electron microscopy shows normal intercalated discs in AngII-treated wild-type mice, while in AngII-treated LIMP-2 KO mice, the intercalated discs have a higher degree of convolution and a higher concentration of adherens junctions (appreciate the dark black spots in the right panel), which is paralleled by the dilated cardiomyopathy in these mice. Bars represent 2 μm. M, mitochondrion; ID, intercalated disc; a, adherens junction; d, desmosome.
) show co-localization of LIMP-2 and cadherin at the ID of cardiac myocytes. Bars represent 50 μm. FIG. 7C, Tissue sections of AngII-treated LIMP-2 knockout and wild-type LVs were immunostained with anti-pan-cadherin. In wild-type mice, the cadherin distribution is confined to the intercalated discs yielding a regular appearance of cadherin, while in LIMP-2 KO mice, the localization of cadherin has lost the typical pattern produced by a strict location at the intercalated disc. Bars represent 250 μm.
FIG. 8: LIMP-2 regulates intercalated disc integrity by regulating the binding of phosphorylated beta-catenin to cadherin. FIG. 8A, Immunoblot (IB) of lysates of neonatal rat cardiac myocytes that were treated either with shRNA against LIMP-2 (shLIMP-2) or with control shRNA. After 10 days of culture, cardiac myocytes show a 92% knockdown of LIMP-2 protein. Equal protein loading was confirmed by GAPDH. FIG. 8B, Immunoblot (IB) shows diminished levels of P-beta-catenin after immunoprecipitation (IP) with anti-pan-cadherin in shLIMP-2 lysates as compared to control lysates. Cadherin loading was comparable in control and shLIMP-2 IP-lysates. Phosphorylation of beta-catenin in total shLIMP-2 and control protein lysates was comparable. *, P=0.0006. FIG. 8C, Immunoblot showing the specificity of the immunoprecipitation with anti-pan-cadherin. When adding IgG instead of pan-cadherin antibody to the protein lysates, no P-beta-catenin is bound.
FIG. 9: FIG. 9A, KLF15 expression assessed by real-time CR in left ventricular biopsies from Ren-2 rats at the age of 10 weeks. After biopsy, rats were allowed to recover and followed to determine whether they would progress to failure or remain compensated. Expression of KLF15 is significantly down-regulated in hypertrophied hearts that remained compensated, but significantly further suppressed in the hypertrophied hearts that quickly progressed to overt failure, indicating that the level of KLF15 suppression identifies failure prone forms of cardiac hypertrophy. FIG. 9B, In situ hybridization for KLF15 in a normotensive control heart compared to hypertrophic myocardium. The widespread nuclear staining in the normal heart is lost in a large number of myocytes, while there is residual staining in non-myocyte nuclei, indicating that KLF15 expression occurs specifically in cardiac myocytes. FIG. 9C, Stable knock-down of KLF15 by lentiviral introduction of short hairpin RNA, induced expression of BNP in cultured NRVM. FIG. 9D is a graph depicting relative MEF2 luciferase activity.
FIG. 10: FIG. 10A, Addition of TGFβ (10 ng/ml) to cultured cardiac myocytes almost completely suppressed KLF15 mRNA expression. Stable knock-down of the TGFβ type I receptor by lentiviral introduction of short hairpin RNA abolished this effect, demonstrating that TGFβ via its TGFβ type I receptor is capable of suppressing KLF15 expression. FIG. 10B, Whole heart homogenate immunoblotted against the Tgfβ type I receptor shows a substantial and significant reduction in expression of Tgfβ type I receptor, but no compete loss of the receptor. FIG. 10C, Immunohistochemistry demonstrates that the myocyte specific activation of cre resulted in a clear and robust loss of TGFβ type I receptors from cardiac mycocytes when comparing WT hearts to the MerCreMer-TGFβ type I mice. FIG. 10D, Angiotensin II infusion induced a significant hypertrophic response in Wt mice, which was blunted in MerCreMer-TGFβ type I mice. FIG. 10E, Angiotensin II infusion induced a significant loss in fractional shortening as an indicator of loss of cardiac function, which was blunted in MerCreMer-TGFβ receptor mice. FIG. 10F, Angiotensin II infusion and subsequent hypertrophy induced a down-regulation of KLF15, which was blunted in the MerCreMer-TGFβ receptor mice.
FIG. 11: The upper panel shows significantly up-regulated KLF15 mRNA in the mouse heart after AAV9-KLF15 injection, compared to green fluorescent protein (AAV9-GFP) injection. **: p<0.05 comp to GFP group. *: p<0.05 compared to GFP group. #: p<0.05 compared to GFP+AngII group. The lower panel shows significantly less hypertrophy in the AAv9-KLF15 group after AngII stimulation compared to the AAV9-GFP group with AngII (#: p<0.05). Statistical analysis with student\'s t-test, n=3-5 animals/group.
DETAILED DESCRIPTION OF THE INVENTION
Lysosomal Integral Membrane Protein-2 is a Novel Component of Intercalated Discs and Prevents Cardiomyopathy
Materials and Methods
Ren-2 Rats, Microarray Analysis and Immunoblotting
From 10-week-old Ren-2 and Sprague-Dawley (SD) rats (Mollegard, Lille Skensveld, Denmark), a biopsy of the LV was taken as described previously (Van Haaften et al., 2006). Rats were followed by serial echocardiography at 10, 12, 15, 16, 18, 19 and 21 weeks of age and sacrificed at 15-18 weeks upon clinical signs of heart failure (heart failure-prone/HF-prone rats) or at 21 weeks when clinical signs of failure had not appeared (compensated/comp rats). Total RNA was isolated from LV biopsies and amplified as previously described (Schroen et al., 2004; Heymans et al., 2005), hybridized to Affymetrix rat 230 2.0 GeneChips and analyzed with Microarray Analysis Suite Software 5.0. LV protein extracts (50 μg) were immunoblotted with polyclonal rabbit anti-LIMP-2 (Novus Biologicals, Littleton, Colo., 1:500) and polyclonal rabbit anti-GAPDH (Abcam, Leusden, Netherlands; 1:10,000).
LIMP-2 Knockout Mice, RNA Isolation and Quantitative PCR Analysis
Ten- to twelve-week-old male LIMP-2 KO and WT C57/B16 mice weighing 20-25 grams were used. To study blood pressure effects of AngII, arterial pressures were monitored during intravenous infusions at doses of 0.5, 1.5, 5, 15, and 50 ng per minute. To study development of LV hypertrophy, AngII (1.5 μg/g/day) was infused subcutaneously by osmotic minipump 2004 (Alzet osmotic pumps, Cupertino, Calif.) for 28 days.
Echocardiography was performed at day 0, day 14 and day 28. At day 28, mice were hemodynamically monitored (dP/dt) using Millar® under basal and dobutamin-stimulated conditions; afterward, LVs were removed. RNA was isolated with RNeasy mini kit (Qiagen, Valencia, Calif.) and SYBR Green quantitative PCR analysis was performed on a BioRad iCycler to determine BNP, ANF and alpha-skeletal actin (aska) expression (Table 1). LV sections were stained with hematoxylin-eosin (HE) and Picro serious red (SR) as described before (Junqueira et al., 1979), or were immunohistochemically stained with monoclonal mouse anti-pan-cadherin (Sigma, Saint Louis, USA; 1:500) and monoclonal mouse anti-human desmin (Dako Cytomation, Denmark, 1:50). Ultrastructural analysis was performed by transmission electron microscopy as described previously (Schroen et al., 2004).
LIMP-2 in Aortic Stenosis and Heart Failure Patients
RNA was isolated from transmural biopsies obtained from 20 aortic stenosis patients and seven non-hypertrophic control patients as described before (Heymans et al., 2005), and SYBR Green quantitative PCR analysis was performed to determine LIMP-2 expression (Table 1).
Double immunofluorescent stainings with rabbit anti-LIMP-2 (1:250, Cy2) and mouse anti-pan-cadherin (1:500, Cy3) were done on sections of one control subject and two patients that died of overt heart failure, as defined by an ejection fraction of less than 35%. Nuclear counterparts were stained with Topro-3 (Invitrogen, Breda, The Netherlands). Sections were imaged with a laser scanning confocal system (Leica, Rijswijk, The Netherlands), digitized at a final magnification of ×126 and analyzed with Leica Confocal Software. The ethics committees of the Academic Hospital Maastricht and of University Hospital Leuven approved the study, and all patients gave informed consent.
Cell Culture and Lentiviral Vector
A rat-LIMP-2 shRNA expressing lentiviral vector was generated by annealing complementary shLIMP-2 oligonucleotides (Table 1) and ligating them into HpaI XhoI digested pLL3.7 puro vector DNA (modified from a kind donation by Luk van Parijs, Massachusetts Institute of Technology, Cambridge, USA). Lentiviral production was performed by co-transfection of 3 μg shLIMP-2/pLL3.7 puro or empty pLL3.7puro and packaging vectors into 293FT cells by Lipofectamine 2000 (Invitrogen) and virus-containing supernatant was harvested after 48 hours.
Rat ventricular cardiac myocytes (RCMs) were isolated by enzymatic disassociation of 1- to 2-day-old neonatal rats as described previously (De Windt et al., 1997). For lentiviral infection, RCMs were plated on gelatinized six-well plates with 5*105 cells per well, cultured overnight in DMEM/M199 (4:1) media supplemented with 10% horse serum, 5% newborn calf serum, glucose, gentamycin and AraC, and next day infected with shLIMP-2 or empty lentivirus, facilitated by Polybrene (Sigma). After puromycin selection (3 μg/ml), infection efficiencies were above 80%. After ten culture days, cellular protein was isolated for immunoprecipitation (IP) with anti-LIMP-2 (1:100), monoclonal mouse anti-pan-cadherin (Sigma, 1:100) or IgG. IP lysates were immunoblotted with monoclonal anti-pan-cadherin (1:5000), polyclonal anti-phospho-beta-catenin (Ser33/37/Thr41; Cell Signaling Technology, Danvers, Mass., USA, 1:1000) and monoclonal anti-beta-catenin (BD Transduction Laboratories, Franklin Lakes, USA, 1:1000).
For stretch experiments, RCMs were cultured on a collagen type I-coated silastic membrane (Specialty Manufacturing, Inc., USA) and subjected to static, equibiaxial stretch during a six-hour period. RNA was isolated with RNeasy mini kit (Qiagen) for LIMP-2 SYBR Green quantitative RT-PCR (Table 1).