The present invention relates to a method of identifying and/or obtaining a compound which is suspected to be an inhibitor or antagonist of H-FABP comprising (i) contacting a host cell which expresses an H-FABP with a compound suspected to be an inhibitor or antagonist of H-FABP under conditions allowing interaction of said compound with said H-FABP ; (ii) determining the activity of said H-FABP ; (iii) determining the activity of H-FABP in a host cell as defined in (i), which has not been contacted with said compound; and (iv) quantitatively relating the activity determined in (ii) and (iii), whereby a decreased activity determined in (ii) in comparison to (iii) is indicative for an inhibitor or antagonist. Furthermore, the invention provides a method of identifying and/or obtaining a compound which is suspected to be an inhibitor or antagonist of an intracellular signaling pathway comprising (i) contacting a host cell which expresses an H-FABP with a compound suspected to be an inhibitor or antagonist of an intracellular signaling pathway under conditions allowing interaction of said compound with said H-FABP; (ii) determining the activity of the intracellular signalling pathway; (iii) determining the activity of the intracellular signalling pathway in a host cell as defined in (i), which has not been contacted with said compound; and (iv) quantitatively relating the activity determined in (ii) and (iii), whereby a decreased activity determined in (ii) in comparison to (iii) is indicative for an inhibitor or antagonist. In addition, the invention relates to a method of refining a compound or a protein identified by the methods described herein. Also described are uses of compounds as identified and/or obtained by the inventive methods in the preparations of pharmaceutical and diagnostic compositions for the treatment or prevention of hypertrophy and/or impaired or increased (3-oxidation. Finally, pharmaceutical and diagnostic compositions are disclosed which comprise the compounds of the present invention.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer's specifications, instructions, etc. ) are hereby incorporated by reference.

Cardiac failure occurs when, despite normal venous pressures, the heart is unable to maintain sufficient cardiac output to meet the demands of the body. The incident of heart failure is estimated at 10 per 1000 over 65 years of age and, irrespective of the aetiology, prognosis is poor.

Cardiac failure comprises a plurality of clinical syndromes which are historically subdivided into syndromes of right, left and biventricular (congestive) cardiac failure, but it is rare for any part of the heart to fail in isolation.

The body has many options to regulate the changing demand in oxygen and nutrients in short term by increased heart rate and blood pressure or in long term by increased contractility or metabolism.

However, upon an insult to the myocardium due to chronic pressure overload, ischemia, infection, toxic or genetic events, the body compensates for the loss of pumping power usually with mechanisms made for short term compensation. The chronic use of these compensatory mechanisms however leads to an overburdening of the heart muscle and finally into decompensation and heart failure.

As far as the congestive heart failure (CHF) and/or the system alterations in heart insufficiencies are concerned, dramatically reduced exercise tolerance and pulmonary edema can be observed. The macroscopic manifestation of disease progression is the initial thickening of the left ventricular myocardium, followed by a severe dilatation of a thin-walled left ventricular cavity. These alterations are reflected on the cellular level by distinct forms of cardiomyocyte hypertrophy. The early phase (adaptive) is characterized by thick cardiomyocytes with parallel organization of sarcomeres while the later, decompensated stage is characterized by elongated cells with the serial sarcomere organization.

15 Mio patients are worldwide afflicted by CHF. The current therapeutic regimen combines treatment of CHF symptoms by means of diuretics with reduction of stress for the diseased heart by lowering blood pressure and if heart failure is not to advanced by reducing adrenergic drive. Yet, current therapy is still inadequate to stop progression or induce reversion of the disease, see e. g. Alexander, Hurt's The Heart, gth edition, Mc Graw Hill, (2000) and there is no causal treatment for CHF available.

It is known that changes in metabolism due to environmental influence or genetic alterations are major risk factors for many diseases, among them the above discussed cardiovascular diseases as well as other common diseases such as obesity or diabetes. The strong link between many of these diseases is reflected in the term metabolic syndrome. However, since metabolism is very complex and a basic feature, therapeutic intervention requires exact molecular understanding of the metabolic changes in an affected organ. The very tight coupling of metabolic feedback networks have to be carefully considered since metabolism is not only catabolic energy generation, but also anaboiic synthesis of important building blocks for all cellular functions. In addition, metabolism is closely linked to the network of signaling pathways. In respect to energy generation, organs have different possibilities to cover their energy demand according to the actual physiologic situation. Major sources of fuel are lipids, carbohydrates and proteins.

However, there are very few reports on the human heart metabolism in health and disease.

Paolisso (1994 in Metabolism 43, 174-179) reported a nearly equimolar myocardial glucose and lipid uptake and oxidation rate (10 pmol/min) for healthy control subjects.

In patients with congestive heart failure a significant shift towards fatty acid oxidation was observed (16 pmoi/min lipids vs 4 pmol/min Glucose).

Lommi (1998 in Am. J. Cardio. 81,45-50) confirmed an increased overall lipid utilization in CHF patients, without specifically addressing the myocardial metabolism.

They calculated in control subjects a lipid oxidation of 30 j/kg*min and a glucose oxidation of. 15 J/kg*min, which reflects a 2: 1 ratio. In CHF patients this ratio was

determined as 2,5 : 1 (37 J/kg*min vs 14J/kg*min), which was mainly explained by an increase of epinephrine levels, which is a potent stimulator of lipoprotein lipase.

The metabolic alterations described by Paolisso, loc. cit, theoretically would lead to an improvement of the energy balance, because the generation of 1,29 mmol ATP/min from fatty acids and 0,36 mmol ATP/min from glucose (ratio 4: 1) in healthy subjects is shifted to 2,06 mmol ATP/min from fatty acids and 0,14 mmol ATP/min from glucose (ratio 16: 1) in CHF patients.

This is in clear contrast to the observed energy depletion in heart failure described by Katz (1989,1990, 1993, 1998), Shen et al. (Circulation, 1999) or Zhang et al. (Circulation 1996) and to the reduction in the expression and activity of major ATP consuming heart proteins such as SR Ca2+ATPase (SERCA) (Meyer et al. 1995) and -Myosin Heavy Chain (Nakao, J. Clin. Invest. 100 (1997), 2362-2370).

In order to explain this paradoxon, one has to consider the complex implications of the changed metabolism. Since the molar ratio of lipids : glucose is shifted from 1: 1 to 4: 1 (Paolisso, 1994, loc. cit. ) the dramatically reduced influx of glucose leads to an increased giuconeogenesis, to provide the cell with the necessary glucose building blocks. This subsequently leads to a deprivation of intermediates for the Krebs cycle, since malate is constantly channeled into gluconeogenesis by the cytosolic malate- DH.

Weinberg, Proc. Natl. Acad. Scie. USA 97 (2000), 2826-2831 described severe energy deficits in tissues after episodes of hypoxia/reoxygenation. This was explained by impaired respiratory complex I activity and subsequent mitochondrial dysfunction. The impairment was attenuated by anaerobic substrate oxidation of a--Ketoglutarate, which however is reduced when Krebs cycle intermediates are depleted.

Since even in non-ischemic CHF a relative hypoxia is very common (due to hypertrophic growth of the heart muscle without increased number of capillaries) it is assumed that increased fatty acid oxidation does not necessarily lead to an improved energy balance of the diseased heart muscle.

Since there is no causal treatment for cardiac failure, in particular for CHF, available, there is a need for means and methods which can lead to the provision of compounds useful in the prevention and or treatment of heart diseases.

The solution to this technical problem is achieved by providing the embodiments characterized in the claims.

Accordingly, the present invention relates to a method of identifying and/or obtaining a compound which is suspected to be an inhibitor or antagonist of H-FABP comprising the steps of (I) contacting a host cell which expresses an H-FABP with a compound suspected to be an inhibitor or antagonist of H-FABP under conditions allowing interaction of said compound with said H-FABP; (II) determining the activity of said H-FABP; (ici) determining the activity of H-FABP in a host cell as defined in (i), which has not been contacted with said compound; and (IV) quantitatively relating the activity determined in (H) and (ici), whereby a decreased activity determined in (II) in comparison to (vil) is indicative for an inhibitor or antagonist.

The term"H-FABP"in accordance with this invention means heart or cardiac fatty acid binding protein. H-FABP is also known to the skilled artisan as FABP3. The FABP3 or H-FABP gene is located on human chromosome 1. The corresponding gene was described to contain 4 exons (Genbank entry U57623, cDNA). The human mRNA for FABP is available under the accession number X56549 and can, inter alia, be found under www. ncbi nim. nih. aov and the coding sequence of FABP3 is shown in SEQ ID NO: 1. Cardiac fatty acid binding protein (H-FABP) has been isolated from heart by a combination of gel filtration and ion exchange chromatography (Fournier1978). Biochem. Biophys. Acta 533,457-464). The primary structure was later determined by cloning of the cDNA (Peeters 1991, Biochem. J. 276,203-207) and found to be identical to that of muscle FABP: The molecular mass of the 132 residue protein is 14727 g/mol ; the theoretical pl is 6.34 and the calculated molar extinction coefficient at 280 nm is 13940 M-'cm-'.

The three-dimensional structure of recombinant H-FABP has been determined by X- ray crystallography at 2.1 A resolution (Zanotti (1992). J. Biol. Chem. 267,18541- 18550). Later on the structure was refined to 1.4 A resolution (Young (1994).

Structure 2,523-534). Also solution structures of bovine (Lassen (1995). Eur. J.

Biochem. 266, 266-280) and human H-FABP (Luecke (2001) 354, 259-266) were

solved. The solved three-dimensional structures proved for means to select potential theoretical inhibitors/antagonists of H-FABP, e. g. structural inhibitors/antagonists, which may be further tested and/or characterized by the inventive method.

H-FABP is the only known FABP isoform in heart and skeletal muscle, and ovaries. Other tissues expressing H-FABP contain at least one more isoform which could reflect partial functional redundancy. The main function of the cytoplasmatic FABPs seems to be the transport of free fatty acids (FFAs) through the hydrophilic cytosol ; for review see Storch and Thumser, Biochem. Biophys. Acta. 1486 (2000), 28-44.

H-FABP employed in the method of the present invention may comprise H-FABP from different species, preferably, human H-FABP is employed. Furthermore, it is envisaged that mutant forms of H-FABP are employed. Most preferred are mutants which lost their affinity for fatty acids (FFAs), but have still other functional properties as described herein. Mutants that lost their ability to interact with FFAs or which show less affinity for FFAs are known in the art, see Zimmerman (1999), 344,495-501. Such mutants to be employed in the inventive method should however still be able to induce H-FABP activities as disclosed herein and as illustrated in the appended examples. These activities comprise, but are not limited to the capability to from a hypertrophic phenotype upon intracellular expression of H-FABP in host cells, preferably in cardiomyocytes, or the capability to interact with signaling pathways as described herein below. It is also envisaged that isoforms and/or splice variants of H- FABP are employed in the inventive method (s). Such isoforms and/or splice variants are described herein and in the appended examples. Most preferably the H-FABP, isoform and/or splice variants are predominantly expressed intracellularly in the method of the invention.

As documented in the appended examples, in the present invention it was surprisingly found that intracellular (over) expression of H-FABP in primary cardiomyocytes leads to a severe/profound hypertrophic effect in said cells with parallel sarcomere organization. Said parallel sarcomere organization is the cellular phenotype occurring in adaptive/concentric myocardial hypertrophy. The surprising finding that intracellular expression of H-FABP leads to a potent induction of (cardiomyocyte) hypertrophy provides for novel and inventive methods for

therapeutic interventions for cardiological disorders, in particular of myocardial diseases and most preferably of congestive heart failure (CHF). This is in particular surprising since in the prior art it was speculated that cardiac hypertrophy can only be induced by H-FABP, when purified, native H-FABP is presented as a ligand to specific, extracellular, high affinity receptors, see Burton (1994) Biochem. Biophys.

Res. Com. 205, 1822-1828. As will be discussed herein below and in the appended examples it was furthermore surprisingly found that H-FABP is capable of specifically inducing cell signaling pathways. These surprising findings provide for means and methods for identifying and/or obtaining inhibitors and/or antagonists of H-FABP which, in turn, may be employed in the prevention and or treatment of heart diseases related to hypertrophic cardiomyocytes, dilated cardiomyopathy (idiopathic), hypertrophic cardiomyopathy, restrictive cardiomyopathy, obstructive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, unclassified cardiomyopathy, ischemic cardiomyopathy, valvular cardiomyopathy, hypertensive cardiomyopathy, inflammatory cardiomyopathy, metabolic cardiomyopathy, infiltrative cardiomyopathy, muscular dystrophy-associated cardiomyopathy, neuromuscular disorder-associated cardiomyopathy, toxic cardiomyopathy, or peripartum cardiomyopathy. The findings disclosed herein are even more surprising in light of the fact that the prior art (see in particular Heinke (1998), Electrophoresis 19, 2021- 2030) has proposed that H-FABP expression is decreased in the failing heart. inhibitors or antagonists as identified and/or obtained by the method of the present invention are particularly useful in the therapeutic management, prevention and or treatment of coronary disease, specifically in diseases like congestive heart failure (CHF), dilative and concentric or ischemic cardiomyopathy. Since many conditions, such as hypertension, arrhythmia, coronary artery diseases, stable and unstable angina pectoris, arteriosclerosis, diabetes, hyperglycemia, hyperinsulinemia, and hyperlipidemia can lead to CHF and all these conditions are related to the metabolic syndrome mentioned herein above, the inhibitors or antagonists as identified and/or obtained by the method of the present invention are also useful for the prevention and/or the treatment of said conditions.

Said inhibitors and/or antagonists which are suspected to be an inhibitor or antagonist of H-FABP and/or its activity is preferably a compound that interacts

and/or interferes with H-FABP. Said interaction may be a direct interaction, like a direct protein/protein or protein/nucleic acid molecule interaction, but it is also envisaged that said interaction may be mediated by further, additional compounds.

Therefore, potential inhibitors or antagonists to be identified, screened for and/or obtained with the method of the present invention include molecules, preferably small molecules which bind to, interfere with and/or occupy relevant sites on H-FABP.

It is furthermore envisaged that such inhibitors interfere with the synthesis/production of functional H-FABP, like, e. g. anti-sense constructs and the like inhibitors and/or antagonist which can be screened for and obtained in accordance with the method of the present invention include, inter alia, peptides, proteins, nucleic acids including cDNA expression libraries, antibodies, small organic compounds, small molecules ligands, PNAs and the like.

Accordingly, the inhibitor and/or antagonist of H-FABP may comprises (an) antibody (ies). Said antibody (ies) may comprise monoclonal antibodies as well as polyclonal antibodies. Furthermore, chimeric antibodies, synthetic antibodies as well as antibody fragments (like Fab, F (ab) 2, Fv, scFV), or a chemically modified derivative of antibodies is envisaged. It is envisaged that said antibodies binds to H- FABP and/or interferes with the activity of H-FABP.

In addition, oligonucleotides and/or aptamers which specifically bind to H-FABP as defined herein or which interfere with the H-FABP activity are envisaged as inhibitors and/or antagonists of H-FABP. The term"oligonucleotide"as used in accordance with the present invention comprises coding and non-coding sequences, it comprises DNA and RNA and/or comprises also any feasible derivative. The term "oligonucleotide"further comprises peptide nucleic acids (PNAs) containing DNA analogs with amide backbone linkages (Nielson, Science 274 (1991), 1497-1500). Oligonucleotides which may inhibit and/or antagonize H-FABP and/or H-FABP activity and which can be identified and/or obtained by the method of the present invention can be, inter alia, easily chemically synthesized using synthesizers which are well known in the art and are commercially available like, e. g. , the ABI 394 DNA- RNA Synthesizers.

In accordance with the present invention, the term aptamer means nucleic acid molecules that can bind to target molecules, preferably to H-FABP as defined herein.

Aptamers commonly comprise RNA, single stranded DNA, modified RNA or modified DNA molecules. The preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sites (Gold, Ann. Rev. Biochem. 64 (1995), 763-797).

As mentioned herein above, said inhibitor and/or antagonist of H-FABBP may also comprise small molecules. These term relates, but is not limited to small peptides, inorganic and/or organic substances or peptide-like molecules, like peptide-analogs comprising D-amino acids.

Furthermore, peptidomimetics and/or computer aided design of appropriate antagonists or inhibitors may be employed in order to obtain candidate compounds to be tested in the inventive method. Appropriate computer systems for the computer aided design of, e. g. , proteins and peptides are described in the prior art, for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N. Y.

Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. The results obtained from the above-described computer analysis can be used in combination with the method of the invention for, e. g. , optimizing known compounds, substances or molecules. Appropriate compounds can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds, e. g. , according to the methods described herein.

Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715. Furthermore, the three-dimensional and/or crystallographic structure of inhibitors or activators of H- FABP or of the nucleic acid molecule encoding for H-FABP can be used for the design of peptidomimetic inhibitors or antagonists H-FABP to be tested in the method of the invention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg.

Med. Chem. 4 (1996), 1545-1558).

The compounds to be screened with the method (s) of the present invention do not only comprise single, isolated compounds. It is also envisaged that mixtures of compounds are screened with the method of the present invention. It is also possible to employ extracts, like, inter alia, cellular extracts from prokaryotic or eukaryotic cells or organisms.

Host cells to be employed in the present invention are disclosed herein below and comprise, preferably cultured cells, even more preferably cardiomyocytes, most preferably primary cardiomyocytes.

As already mentioned herein above, the term"interaction of said compound with H- FABP"as used in accordance with the present invention does not only comprise a direct interaction of said compound with H-FABP but also comprises interaction (s) mediated by further compounds/molecules, for example in signaling pathways or in complex formations.

The term"quantitatively relating"as employed herein above means that the activity of H-FABP which is determined in steps (and (iii) of the inventive method are compared. It is, inter alia, envisaged that the activity (ies) obtained when the compound to be identified and/or obtained by the inventive method is compared to the activity (ies) of a control which comprises the same experimental setting but wherein said compound is omitted. Said"quantitative relation"may be carried out by methods known to the skilled artisan and described herein and in the appended examples,. e. g. morphometric analysis, comparisons of protein patterns, phosphorylation status, etc. Furthermore, it is envisaged that said comparisons and/or quantitative relation are carried out in a computer-assisted fashion. Said comparison/quantitative relation may also comprise the analysis in high-throughput screens.

In a preferred embodiment of the method of the invention, the activity of said H-FABP comprises activation of an intracellular signaling pathway and may comprise the formation of hypertrophy.

As inter alia documented in the appended examples, said hypertrophy, e. g. the hypertrophic phenotype or the formation of a hypertrophic phenotype may be measured by morphometric analysis. Further methods comprise, but are not limited to fluorescence studies as also documented in the appended examples, measurements of protein content of a cell in comparison to a non-hypertrophic cell, activation of hypertrophy associated signaling cascades, or gene expression programs (reporter gene assays). Moreover the secretion of cardiac natriuretic peptides (Atrial Natriuretic Peptides = ANP and Brain Natriuretic Peptides = BNP) is also comprised. Said peptides are synthesized and secreted by the heart, producing several biological effects, such as natriuresis, vasorelaxation, hypertension and neuromodulation. ANP and BNP plasma concentrations have been shown to be markedly increased in patients with acute myocardial infarction. Increased expression of ANP/BNP is known to be a marker for heart hypertrophy.

Therefore, in a preferred embodiment of the present invention, the inventive method also relates to a method, wherein the activity of said H-FABP comprises increasing or decreasing the amount or changing the subcellular localization of a cellular protein indicative for hypertrophy. Such proteins, which are indicative for hypertrophy are naturally occurring proteins like, DCMAG-1 (see WO 98/56907), MHC (ßMHC, as described in Lowes, J. Clin. Invest. 100 (1997), 2315-2324), PKCJ3 (as described in Bowling, 1999, Circulation 99,334-337) or PKB/Akt (as described herein below and in Shioi, EMBO J. 19 (2000), 2537-2548), which are up-regulated in cardiac hypertrophy or proteins like oc. MHC (Lowes et ai., J Clin Invest. 100 (1997), 2315- 2324) or Serca2a (Qi et al. Am J Physio. 272 (1997) 5 Pt 2, H2416-H2424 or Zarain- Herzberg et al. Mol Cell Biochem. (1996) 163-164, 285-290), which are down- regulated. Furthermore, it is also envisaged in accordance with this invention, that biochemical modifications are measured in order to identify an hypertrophic phenotype, for example the amount of phosphorylated protein may be determined by measuring the expression level of a reporter gene, the uptake of amino acids and/or the increase in volume. Methods for the determination of hypertrophic phenotypes are known in the art and illustrated in the appended examples.

As shown in the appended examples, the over-expression of H-FABP in cardiomyocytes surprisingly leads to a specific activation of signal transduction

pathways and in particular to an activation of pathways comprising AKT-kinases.

Therefore, it was surprisingly found that over-expression of FABP activates protein kinase B (PKB/Akt) and it is furthermore suggested that the over-expression and/or acitivity of specific markers which are associated with the development of a myocardial hypertrophic phenotype (like PKCß, DCMAG-1, MHC and PKB/Akt) is increased upon FABP over-expression.

It is preferred that in the inventive method, the host cell expresses a reporter gene indicative for hypertrophy and that, furthermore, the cellular protein is encoded by said reporter gene. Said reporter gene may be selected from the group consisting of S-MHC, DCMAG-1, PKB and PKCß. It is further envisaged that marker/reporter genes be employed which are selected form the group consisting of CFP, GFP, YFP, BFP, Firefly luciferase, Renilla luciferase, LacZ, SF=AP and hGH. These marker/reporter genes may be fused/linked to further proteins/peptides. Within the inventive methods further techniques may be employed, inter alia, the expression of said cellular protein and/or reporter gene by (recombinant) viral systems, as well as the measurement of said reporter gene-product in living cells, cell-supernatants and/or cellular extracts.

Marker/reporter genes for signaling pathways are well known in the art, as inter alia documented in Shepherd (1998), Biochem J. 333,471-490 or in Toker (2000). Mol.

Pharmacol. 57,652-658, describing specific signalling cascades.

It is envisaged that these reporter genes are, inter alia, transfected into the host cells.

Furthermore, said reporter genes may be comprised in the genome of said host cells and may be incorporated in said genome by methods known in the art, like, e. g. knock-ins, as described herein below.

The present invention also relates to a method of identifying and/or obtaining a compound which is suspected to be an inhibitor or antagonist of an intracellular signaling pathway comprising (t) contacting a host cell which expresses an H-FABP with a compound suspected to be an inhibitor or antagonist of an intracellular signaling pathway under conditions allowing interaction of said compound with said H-FABP; (ii) determining the activity of the intracellular signaling pathway; (iii)

determining the activity of the intracellular signalling pathway in a host cell as defined in (i), which has not been contacted with said compound; and (iv) quantitatively relating the activity determined in (ii) and (iii), whereby a decreased activity determined in (ii) in comparison to (iii) is indicative for an inhibitor or antagonist.

The corresponding activities may be measured by methods known in the art and comprise but are not limited to kinase assays, phosphorylation/dephosphorylation assays, multimerization/dimerization assays, nuclear translocation assays, reporter- gene assays with specific cis-acting elements, assays measuring the production of "second messengers", like, inter alia, cAMP, cGMP or Ca It is particularly preferred that the above mentioned intracellular signaling pathway comprises Protein kinase B (PKB) /Akt and/or Protein kinase Cß (PKCß) and/or that said activity of an intracellular signalling pathway is Protein kinase B (PKB)/Akt and/or Protein kinase Cß (PKCß) activity.

Within the present invention it is also envisaged that novel compounds be identified and/or be obtained which are of high relevance in the treatment as well in the prevention of heart insufficiency (ies). The methods for identifying as well as validating such compounds as described herein may also be combined. It is, e. g., envisaged that in said activity assays/methods hypertrophic markermolecules are measured upon intracellular expression of H-FABP. Similarly, the stimulation/activation of signaling pathways, in particular the activation of AKT-kinase or PKC can be measured. As mentioned herein above the compound to be tested may lead to a modified activity of H-FABP, wherein, for example a reduced activation of AKT-kinases or PKCJ3 or a reduced expression of hypertrophic markers disclosed herein are indicative for an inhibitor or antagonist of H-FABP/of H-FABP activity.

In addition the present invention provides for a method of identifying and/or obtaining a compound which is suspected to be an inhibitor or antagonist of formation of hypertrophy comprising (i) contacting a host cell which expresses an H-FABP with a compound suspected to be an inhibitor or antagonist of hypertrophic cell morphology under conditions allowing interaction of said compound with said H-FABP; (ii) determining the amount of hypertrophy; (iii) determining the amount of hypertrophy in

a host cell as defined in (i), which has not been contacted with said compound; and (iv) quantitatively relating the activity determined in (ii) and (iii), whereby a decreased amount of hypertrophy in (ii) in comparison to (iii) is indicative for an inhibitor or antagonist.

It is preferred that the amount and/or subcellular localization of marker proteins for hypertrophy (e. g. DCMAG-1/Akt) is determined. Said hypertrophy may be determined by microscopical and/or morphometric means.

As mentioned herein above, such measuring methods are known in the art and comprise biochemical, morphometric as well as morphological (microscopical) analysis and such methods are exemplified in the appended examples.

Preferably, in the inventive method described herein, the H-FABP is selected from the group consisting of (a) a protein comprising an amino acid sequence of SEQ ID NO: 2,4 or 6; (b) a protein which comprises an amino acid sequence which is at least 65% identical to the amino acid sequence of the protein of (a); (c) a protein encoded by a nucleic acid molecule of SEQ ID NO: 1,3 or 5; (d) a protein which comprises in its amino acid sequence at least one conservative amino acid exchange in comparison to the amino acid sequence of the protein of any one of (a) to (c); (e) a protein which comprises at least a functional domain of the protein of any one of (a) to (d).

In accordance with the present invention, a H-FABP protein as described by Peeters (1991), loc. cit. and depicted in SEQ ID NO: 2 may be employed for the inventive method. However, (splice) variants/isoforms of H-FABP may be employed in the here described method.

Preferably, said (splice) variants/isoforms comprise an amino acid sequence as depicted in SEQ ID NOs: 4 or 6 or comprise at least the functional domain (s) as described herein below. Preferably, the protein to be used in the present invention comprises an amino acid molecule which is at least 65%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least

99% identical to the H-FABP proteins described herein and as shown, in particular, in SEQ ID NOs: 2,4 and 6.

The term"functional domain"and"functional domain"of H-FABP means in context of this invention that the H-FABP and its isoforms/variants described herein comprises at least one, preferably at least two, more preferably at least three of the functional activities of H-FABP. These activities comprise activities as known in the art, like FFAs-binding, but, preferably, these activities comprise the capability of H-FABP to the formation of an hypertrophic phenotype and/or the activation of signaling pathway, in particular the activation of PKB/Akt or PKCß. Said hypertrophy caused by the above mentioned hormones could be induced by activating the hypertrophic function of FABP, at least partly. This means that the inhibition of FABP would lead to a decreased or even completely inhibited progress of the cytokine dependent hypertrophy. The term"functional fragment"of H-FABP and its variants/isoforms as employed in the present invention aiso relates to the activities of H-FABP as described herein. Methods for detection and/or verification of the said function of said domain are disclosed herein and in the appended examples.

In a preferred embodiment, said H-FABP further comprises a tag. Said tag may be selected from the group consisting of: His-tag, Flag-tag, Myc-tag, HA-tag, GST-tag, T100, VSV-G, V5, S-tag, HSV, CFP, RFP, YFP, GFP, BFP, Cellulose binding domain (CBD), Maltose binding protein (MBP), NusA-tag, thioredoxin (Trx), DsbA, DabC and a biotinylation sequence. Yet, further tags known by the person skilled in the art are envisaged in context of this invention.

It is most preferred that the host cell employed in the inventive method described herein comprises a recombinant DNA molecule which comprises a DNA encoding a protein as defined herein above, e. g. an H-FABP as described herein with or without tag and/or any further modification.

As also documented in the appended examples, said recombinant DNA molecule is, preferably, a viral vector. Furthermore, said recombinant DNA-moiecufe may be a non-viral vector, a recombinant adeno-viral DNA, a (recombinant) Herpes simplex-

viral DNA, a (recombinant) adeno-associated-viral (AAV) DNA, BACs, PACs, or YACs.

The host cell employed in the inventive method may be stimulated by biochemical, chemical or physiological means. Preferably, and as shown in the appended examples, said host cell is stimulated by LIF. Yet, it is also envisaged and within the scope of the invention, that said host cell is stimulated by hormones like ET-1, ET-2, ET-3, angiotensine I and 11, adrenaline and noradrenaline, insulin, IGF, myotrophin, hormone analoga, like isoproterenol (ISO), phenylephrine (PE), phorbolester and/or cytokines, like LIF, cardiotrophin-1 (CT-1), interleukin-6 and interleukin-11, oncostatin M, ciliary neurotrophic factor. Said stimulation may lead to a hypertrophy.

The host cell employed in the present invention is preferably a mammalian host.

Most preferred is a host cell which is derived from primary cardiomyocytes (pCMs).

However, further host cells may be employed in the inventive method. These cells comprise, but are not limited to, HEK-, Hela-, 3T3-, L-, Jurkat-, COS-, BHK-or CHO- cells. Said cells are cells derived from striated muscle or heart-muscle.

As mentioned herein, it is not only envisaged that in the inventive method tissue-or cell culture systems be employed, but also in vivo test/assay systems are within the scope of the present invention. In vivo and in vitro systems are well known in the art and comprise, inter alia, the production and use of non-human transgenic animals expressing a H-FABP as defined herein (either mutated or non-mutated), as well as corresponding tissue-and/or cell culture systems. Yet, it is particulary preferred that the H-FABP as defined be employed in in vitro systems which also comprise high- throughput screens.

Therefore. it is understood that the inventive method also comprises the use on non- human transgenic animals and cells, organs or tissues thereof. It is, inter alia, envisaged, that non-human transgenic animals be generated which overexpress FABP, preferably H-FABP. This H-FABP may comprise the species-specific H-FABP of said transgenic animal, but it is also envisaged that said animal expresses (overexpress) H-FABP from another species, e. g. human H-FABP. The inhibitor/antagonist of H-FABP and/or its activities can than be tested, screened in

said non-transgenic animals or the inventive method can be employed in isolated cells or organs from these transgenic animals. Preferred non-human transgenic animals are mice, rats, sheep, cows. Yet, other transgenic animals comprising muscle cells, in particular heart muscle cells or equivalents, are envisaged.

The present invention also provides for a method for identifying a protein or a plurality of proteins in heart tissue whose activity is modulated by H-FABP comprising the steps of (I) providing H-FABP or a functional fragment thereof; and (II) identifying a protein or plurality of proteins capable of interacting with H-FABP.

Proteins and or plurality of proteins capable of interacting with H-FABP may be further tested for their ability to function as inhibitors and/or antagonists of H-FABP by the inventive methods described herein above. In addition, said interacting protein (s) could act as activator (s) or modulator (s) of H-FABP-signaling function. All methods, uses and assays described herein are not only useful for scientific purposes but are in particular useful in drug screening methods and for pharmaceutical/pharmacological tests and assays.

The person skilled in the art may identify proteins and/or a plurality of proteins capable of interacting with H-FABP by methods known in the art, also comprising "knock-in"assays. Transfection or infection assays as exemplified in the appended examples. As described herein below, it is particularly preferred in method of the present invention that vectors be employed which encode for H-FABP as described herein. Such vector systems are known in the art and described herein below. For example, it is envisaged that viral system are employed, like retroviral or adenoviral systems. Preferably these are recombinant viral systems, as, inter alia, for adenoviral systems described in He (1998) PNAS 95,2509-2514.

Said"knock-in"assays may comprise"knock-in"in tissue culture cells, as well as in (transgenic) animais. Examples for successful"knock-ins"are known in the art (see, inter alia, Tanaka, J. Neurobiol. 41 (1999), 524-539 or Monroe, Immunity 11 (1999), 201-212). Furthermore, biochemical assays may be employed which comprise, but are not limited to, binding of the H-FABP (or (a) functional fragment (s) thereof) to other molecules/ (poly) peptides and assaying said interactions by, inter alia,

scintillation proximity assay (SPA) or homogenous time-resolved fluorescence assay (HTRFA).

Further interaction assays to be employed in the method disclosed herein may comprise FRET-assays (fluorescence resonance energy transfer; as described, inter alia, in Ng, Science 283 (1999), 2085-2089 or Ubarretxena-Belandia, Biochem. 38 (1999), 7398-7405), TR-FRETs and biochemical assays as disclosed herein.

Furthermore, commercial assays like"Amplified Luminescent Proximity Homogenous Assay" (BioSignal Packard) may be employed. Further methods are well known in the art and, inter alia, described in Fernandez, Curr. Opin. Chem. Biol. 2 (1998), 547- 603.

The"test for interaction"may also be carried out by specific immunological and/or biochemical assays which are wet) known in the art and which comprise, e. g. , homogenous and heterogeneous assays as described herein below.

Said interaction assays employing read-out systems are well known in the art and comprise, inter alia, two hybrid screenings (as, described, inter alia, in EP-0 963 376, WO 98/25947, WO 00/02911; and as exemplified in the appended examples), GST- pull-down columns, co-precipitation assays from cell extracts as described, inter alia, in Kasus-Jacobi, Oncogene 19 (2000), 2052-2059,"interaction-trap"systems (as described, inter alia, in US 6,004, 746) expression cloning (e. g. lamda gtil), phage display (as described, inter alia, in US 5,541, 109), in vitro binding assays and the like. Further interaction assay methods and corresponding read out systems are, inter alia, described in US 5,525, 490, WO 99/51741, WO 00/17221, WO 00114271 or WO 00/05410. Vidal and Legrain (1999) in Nucleic Acids Research 27,919-929 describe, review and summarize further interaction assays known in the art which may be employed in accordance with the present invention. These assays comprise n-hybrid systems.

Homogeneous (interaction) assays comprise assays wherein the binding partners remain in solution and comprise assays, like agglutination assays. Heterogeneous assays comprise assays like, inter alia, immuno assays, for example, ELISAs, RIAs, IRMAs, FlAs, CLIAs or ECLs.

According to a preferred embodiment of the method of the invention said method further comprising the steps of refining the compound or protein identified by the method comprising the steps of: (i) identification of the binding sites of the compound and H-FABP by site-directed mutagenesis or chimeric protein studies; (ii) molecular modeling of both the binding site of the compound and the binding site of H-FABP; and (iii) modification of the compound to improve its binding specificity H-FABP.

Site-directed mutagenesis is a method well known by the skilled artisan and, inter alia, described Sambrook et al., Molecular Cloning : A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988.

It is furthermore preferred that the compound identified and/or obtained by the method, of the invention is further refined by peptidomimetics. Accordingly, in one embodiment the invention relates to a method further comprising the step of refining the compound by peptidomimetics. Appropriate compounds can also be identified as well as refined by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds, e. g., according to the methods described herein. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med.

Chem. 4 (1996), 709-715.

In addition, according to a further preferred embodiment of the invention, the method further comprises the step of modifying the compound identified or refined by the inventive method as a lead compound to achieve, modified site of action, spectrum of activity, organ specificity, and/or improved potency, and/or decreased toxicity (improved therapeutic index), and/or decreased side effects, and/or modified onset of therapeutic action, duration of effect, and/or modified pharmakinetic parameters (resorption, distribution, metabolism and excretion), and/or modified physico- chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or improved general specificity, organ/tissue specificity, and/or optimized application form and route may be modified by esterification of carboxyl groups, or esterification of hydroxyl groups with carbon acids, or esterification of hydroxyl groups to, e. g. phosphates, pyrophosphates or sulfates or hemi succinates, or formation of pharmaceutically acceptable salts, or formation of pharmaceutical

acceptable complexes, or synthesis of pharmacologically active polymers, or introduction of hydrophilic moieties, or introduction/exchange of substituents on aromates or side chains, change of substituent pattern, or modification by introduction of isosteric or bioisosteric moieties, or synthesis of homologous compounds, or introduction of branched side chains, or conversion of alkyl substituents to cyclic analogues, or derivatisation of hydroxyl group to ketales, acetales, or N-acetylation to amides, phenylcarbamates, or synthesis of Mannich bases, imines, or transformation of ketones or aldehydes to Schiffs bases, oximes, acetales, ketales, enolesters, oxazolidines, thiozolidines or combinations thereof.

The present invention also provides a method for the production of a pharmaceutical composition comprising the steps of the method the invention as described herein above and the further step of formulating the compound identified, obtained, refined and/or modified in a pharmaceutical acceptable carrier or diluent.

Therefore, the present invention also relates to a pharmaceutical composition comprising a compound which is obtainable by the method described herein It is, in this context, particularly preferred that a pharmaceutical composition of the present invention comprises an inhibitor and/or antagonist of H-FABP and/or an inhibitor and/or antagonist of the H-FABP activity as described herein said inhibitor/antagonist may inhibit the formation of hypertrophy and/or inhibit/influence the signaling pathways as described herein. Preferably said inhibitor/antagonist of H- FABP and its activity/activities as described and disclosed herein does not interfere with the binding of H-FABP to free fatty acids (FFAs). Yet, it is furthermore envisaged that, optionally, said inhibitor/antagonists modifies/interferes with/inhibits the FFAs binding capacity of H-FABP.

It is also envisaged that the present invention provides for the use of a compound which is obtainable by the method as described herein for the preparation of a pharmaceutical composition for treating a disease related to hypertrophy.

Said disease relating to hypertrophy comprises but is not limited to CHF, dilated cardiomyopathy (idiopathic), hypertrophic cardiomyopathy, restrictive cardiomyopathy, obstructive cardiomyopathy, arrhythmogenic right ventricular

cardiomyopathy, unclassified cardiomyopathy, ischemic cardiomyopathy, valvular cardiomyopathy, hypertensive cardiomyopathy, inflammatory cardiomyopathy, metabolic cardiomyopathy, infiltrative cardiomyopathy, muscular dystrophy- associated cardiomyopathy, neuromuscular disorder-associated cardiomyopathy, toxic cardiomyopathy or peripartum cardiomyopathy. Besides CHF and the several forms of cardiomyopathy mentioned herein above, said disease relating to hypertrophy comprises furthermore conditions which can lead to hypertrophy, e. g. hypertension, arrhythmia, coronary artery diseases, stable and unstable angina pectoris, arteriosclerosis, diabetes, hyperglycemia, hyperinsulinemia and hyperlipidemia.

In particular, the invention relates to the use of a compound which is obtainable by the inventive method described herein for the preparation of a pharmaceutical composition for treating a disease related to impaired or increased p-oxidation. It is envisaged that the inhibitor/antagonist of H-FABP as identified and/or obtained by the inventive method also inhibits-oxidation in cardiomyocytes.

Diseases, which are related to increased ß-Oxidation comprise, inter alia, ketoacidose. The increased production of acetyl-CoA leads to ketone body production that exceeds the ability of peripheral tissue to oxidize them. Ketone bodies are relatively strong acids (pKa around 3.5), and their increase lowers the pH of the blood. This acidification of the blood is dangerous chiefly because it impairs the ability of hemoglobin to bind oxygen. Furthermore the inhibited p-oxidation could be beneficial for type I and type 11 diabetes (skeletal muscle) and could inhibit tumor growth.

It is furthermore envisaged that a protein as defined herein above, i. e. a H-FABP as defined herein, a functional fragment thereof be used for the preparation of a diagnostic composition for diagnosing a disease or a predisposition for a disease related to hypertrophy. It is. also envisaged that a nucleic acid molecule encoding said protein be employed in diagnostic methods. It is particularly preferred that said proteins and/or nucleic acid molecules be employed for the preparation of a diagnostic composition for diagnosing a disease or a predisposition for a disease related to impaired or increased p-oxidation. Said diagnostic composition may be

employed, inter alia, for measuring serum markers which are directly or indirectly related to the level of H-FABP as defined herein.

In a most preferred embodiment, the disease to be treated or diagnosed is congestive heart failure (CHF).

The present invention also provides for a polynucleotide comprising a polynucleotide selected from the group consisting of (a) a polynucleotide having a nucleic acid sequence as shown in SEQ ID NO : 3 or 5; (b) a polynucleotide encoding a polypeptide having an amino acid sequence as shown in SEQ ID NO : 4,6, 8, or 10; (c) a polynucleotide which comprises a fragment encoding at least a specific functional domain of the polypeptide encoded by the polynucleotide of (a) or (b); (d) a polynucleotide which is more than 64.99% identical to a nucleic acid sequence as shown in SEQ ID NO: 3 or encoding a poiypeptide having an amino acid sequence as shown in SEQ ID NO: 4, or a polynucleotide which is more than 91. 11% identical to the polynucleotide having the sequence as shown in SEQ ID NO: 5 or encoding a poiypeptide having an amino acid sequence as shown in SEC2 ID NO: 6, or a polynucleotide which is at least 65% identical to a nucleic acid sequence encoding a polypeptide having an amino acid sequence as shown in SEQ ID NO: 8 or 10 ; and (e) a polynucleotide which hybridizes with the polynucleotide of any one of (a) to (d), wherein said poiynucleotide encodes a polypeptide having the biological function of a polypeptide encoded by the polynucleotide of any one of (a) to (d).

Preferably said polynucleotide which is more than 64.99% identical to a nucleic acid sequence as shown in SEQ ID NO: 3 or encoding a polypeptide having an amino acid sequence as shown in SEQ ID NO: 4 is more than 65%, more preferably more than 70%, even more preferably more than 80%, still more preferably more than 90% and most preferably more than 99% identical to said sequences.

Furthermore, said polynucleotide which is more than 91. 19 % identical to a nucleic acid sequence as shown in SEQ ID NO: 5 or encoding a polypeptide having an amino acid sequence as shown in SEQ ID NO: 6 is preferably more than 91.2%, more preferably more than 92%, even more preferably more than 95% and most preferably more than 99% identical to said sequences,

Said polynucleotide which is at least 65% identical to a nucleic acid sequence encoding a polypeptide having an amino acid sequence as shown in SEQ ID NO: 8 or 10 is preferably at least 70%, especially at least 80%, advantageously at least 90% and most preferably at least 99% identical to said sequences.

In the context of the present invention, the inventive polynucleotide may also be a polynucleotide which comprises a nucleic acid sequence as shown in SEQ ID NOs: 3,5, 7 or 9. As documented in the appended examples, the present invention also provides for novel isoforms of H-FABP, which are in particular useful in the method of the present invention.

These isoforms/variants comprise, in contrast to the H-FABP as described by Peeters et al. (1991), loc. cit. , in their encoding polynucleotide sequences additional polynucleotides leading to novel partial coding sequences as shown in SEQ ID NOs : 7 and 9. The transcription of said novel, additional polynucleotide-isoforms leads to H-FASP molecules with truncated C-terminal parts. As documented in Prinsen (1996), Biochem. J. 315,253-260, such a truncation may lead to a reduced or no fatty acid-binding.

In accordance with the present invention, the polynucleotide of the present invention is at least 65%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, most preferably at least 99% identical to the polynucieotides as depicted in SEQ ID NO : 3. The invention also relates to polynucleotides which are at least 95% identical to the SEQ ID NO: as shown in SEQ ID NO: 5. Identities to the polynucleotide sequences may be deduced by standard programs, like NCBI-biast searches.

The term"functional domain"as employed herein above has the meaning as already defined in context of the inventive method disclosed herein above. In particular, the polypeptide encoded by a polynucleotide disclosed herein above (or any fragment or functional domain thereof) is capable of elucidating a hypertrophic phenotype as described in the appended examples and/or is capable of activating a signaling pathway, in particular the activation of PKB/Akt or PKC.

Furthermore, the invention provides for a vector comprising the polynucleotide of described herein above.

The vector of the present invention may be, e. g. , a plasmid, cosmid, virus, bacteriophage or another vector used e. g. conventionally in genetic engineering, and may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.

Furthermore, the vector of the present invention may, in addition to the nucleic acid sequences of the invention, comprise expression control elements, allowing proper expression of the coding regions in suitable hosts. Such control elements are known to the artisan and may include a promoter, a splice cassette, translation initiation codon, translation and insertion site for introducing an insert into the vector.

Preferably, the nucleic acid molecule of the invention is, operatively linked to said expression control sequences allowing expression in eukaryotic or prokaryotic cells.

Furthermore, inventive nucleic acids can be employed in recombinants or vectors or viruses, inventive (poly) peptides can be expressed by or in recombinants or vectors or viruses and recombinants or vectors or viruses of the invention can be generated and employed as in or in a manner analogous to the methods for making and/or using and/or administering a vector, either in vivo or in vitro, see e. g. , U. S. Patent Nos. 4,603, 112,4, 769,330, 5,174, 993,5, 505, 941, 5,338, 683,5, 494,807, 4,722, 848, 5,942, 335,5, 364,773, 5,762, 938, 5,770, 212,5, 942,235, 5, 756, 103,5, 766, 599, 6,004, 777,5, 990, 091,6, 033,904, 5,869, 312,5, 382,425, WO 94/16716 or WO 96/39491.

Control elements ensuring expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As mentioned herein above, they usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptionai as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulator elements permitting expression in for example mammalian host cells comprise the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rous sarcoma virus), human elongation factor la-promoter, aPM-I promoter (Schaffer, Biochem. Biophys.

Res. Commun. 260 (1999), 416-425), or inducible promoter (s), like, metallothionein or tetracyclin, or enhancers, like CMV enhancer or SV40-enhancer. For the expression in prokaryotic cells, a multitude of promoters including, for example, the tac-lac-promoter or the trp promoter, has been described. Besides elements which are responsible for the initiation of transcription such regulator elements may also comprise transcription termination signals, such as SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pSPORT1 (GIBCO BRL), Casper, Casper-HS43, pUAST, or prokaryotic expression vectors, such as lambda gt11. Beside the nucleic acid molecules described herein above and comprising, inter alia, splice variants of the H-FABP, the vector may further comprise nucleic acid sequences encoding for additional signals, like secretion, signals. Such sequences are well known to the person skilled in the art. Furthermore, depending on the expression system used leader sequences capable of directing the (poly) peptide to a cellular compartment may be added to the coding sequence of the nucleic acid molecules of the invention and are well known in the art. The leader sequence (s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a protein thereof, into the periplasmic space or extracellular medium. Optionaily, the heterologous sequence can encode a fusionprotein including a C-or N-terminal identification peptide imparting desired characteristics, e. g., stabilization or simplified purification of expressed recombinant product. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the. nucleotide sequences, and, as desired, the collection and purification of the (poly) peptide (s) or fragments thereof of the invention may follow.

Furthermore, the vector of the present invention may also be a gene transfer or gene targeting vector. These vectors are also useful in the methods described herein above and may be employed to express/over-express H-FABP and/or the isoforms described herein in host cells which may be employed in the inventive methods.

Gene therapy, which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Preferably the disclosed novel isoforms/splice variants of H-FABP may be employed in the prevention and/or treatment of genetic disorders, like hyperlipidemia, diabetes (preferably diabetes type 11), or deficiencies of carnitine palmitoyl transgerase-1 (CPT-I), carnitine palmitoyl transgerase-2 (CPT-2) or carnitine acyltransferase (CAT).

Suitable vectors, methods or gene-delivering systems for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e. g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086 ; Onodua, Blood 91 (1998), 30-36; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-2251 ; Verma, Nature 389 (1997), 239-242; Anderson, Nature 392 (Supp. 1998), 25-30; Wang, Gene Therapy 4 (1997), 393-400; Wang, Nature Medicine 2 (1996), 714-716 ; WO 94/29469 ; WO 97/00957 ; US 5,580, 859; US 5,589, 466; US 4,394, 448 or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and references cited therein.

In particular, said vectors and/or gene delivery systems are also described in gene therapy approaches in cardiological research, for example in Miyamoto, Proc. Nat). Acad. Sci. USA 97 (2000), 793-798. The nucleic acid molecules and vectors of the invention may be designed for direct introduction or for introduction via liposomes, viral vectors (e. g. adenoviral, retroviral, lentiviral), electroporation, ballistic (e. g. gene gun) or other delivery systems into the cell. Additionally, a baculoviral system can be used as eukaryotic expression system for the nucieic. acid molecules of the invention.

The present invention also provides for a host cell comprising the inventive polynucleotide or the vector of the invention. Such host cells comprise eukaryotic as well as prokaryotic cells as described herein.

The present invention also relates to a host cell transfected or transformed with the vector of the invention or a non-human host carrying the vector of the present invention, i. e. to a host cell or host which is genetically modified with a nucleic acid molecule according to the invention or with a vector comprising such a nucleic acid

molecule. The term"genetically modified"means that the host cell or host comprises in addition to its natural genome a nucleic acid molecule or vector according to the invention which was introduced into the cell or host or into one of its predecessors/parents. The nucleic acid molecule or vector may be present in the genetically modified host cell or host either as an independent molecule outside the genome, preferably as a molecule which is capable of replication, or it may be stably integrated into the genome of the host cell or host.

The host cell of the present invention may be any prokaryotic or eukaryotic cell. Suitable prokaryotic cells are those generally used for cloning like E. coli or Bacillus subtilis. Furthermore, eukaryotic cells comprise, for example, fungal or animal cells. Examples for suitable fungal cells are yeast cells, preferably those of the genus Saccharomyces and most preferably those of the species Saccharomyces cerevisiae. Suitable animal cells are, for instance, insect cells, vertebrate cells, preferably mammalian cells, such as e. g. CHO, Hela, NIH3T3 or MOLT-4. Further suitable cell lines known in the art are obtainable from cell line depositories, like the American Type Culture Collection (ATCC).

In a more preferred embodiment the host cell which is transformed with the vector of the invention is a cardiomyocyte, preferably a primary cardiomyocyte. Yet, further hosts/host cells are envisaged and comprise in vitro differentiated muscle cells, preferably heart muscle cells, which are preferably derived from (mammalian) stem cells. Said stem cells may be adult, preferably, embryonic stem ceils. These mammalian cells may be derived from species like mouse, pig, cow, sheep, dog, monkeys or apes. For certain experimental and/or medical approaches, also human cells are envisaged.

Hosts may be non-human mammals, most preferably mice, rats, sheep, calves, dogs, monkeys or apes. Said mammals may be indispensable for developing a cure, preferably a cure for cardiological disease, in particular for CHF. Furthermore, the hosts of the present invention may be partially useful in producing the (poly) peptides (or fragments thereof), of the invention. It is envisaged that said (poly) peptide (or fragments thereof), like specific splice-variants of H-FABP as disclosed herein be isolated from said host.

The present invention also envisages non-human transgenic animals comprising a mutated form of the nucleic acid molecules of the invention, for example a mutated splice variant of H-FABP, or non-human transgenic animals wherein the nucleic acid molecule of the present invention has been deleted and/or inactivated. Said deletion may be a partial deletion.

Furthermore, the present invention relates to a method of producing a (poly) peptide encoded by the nucleic acid molecule of the invention comprising culturing the host cell of the present invention under suitable conditions that allow the synthesis of said (poly) peptide and recovering and/or isolating the (poly) peptide produced from the culture.

The transformed hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth. The (poly) peptide of the invention can then be isolated from the growth medium, cellular lysats, cellular membrane fractions or inclusion bodies. Once expressed, the protein of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel eiectrophoresis and the like ; see, Scopes, "Protein Purification", Springer-Verlag, N. Y. (1982). Substantially pure proteins of at least about 60%, at least about 70%, at least about 80% or at least about 90 to 97% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred, for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the proteins may then be used therapeutical (including extracorporeally) or in developing and performing assay procedures.

Additionally, the present invention relates to a polypeptide encoded by the polynucleotide described herein above or produced by or obtainable by the above- described method. The term"polypeptide"as employed herein denotes either a peptide, a full-length protein or (a) fragment (s) thereof. A peptide is preferably a fragment of the (poly) peptide of the invention. The term"polypeptide comprises (a) peptide (s) or (a) (poly) peptide (s) which encompass amino acid chains of any length, wherein the amino acid residues are linked by covalent peptide bonds.

The (poly) peptides of the present invention may be recombinant (poly) peptides expressed in host cells like bacteria, yeasts, or other eukaryotic cells, like mammalian or insect cells. Alternatively, they may be isolated from viral preparations. In another embodiment of the present invention, synthetic (poly) peptides may be. used. Therefore, such a (poly) peptide may be a (poly) peptide as encoded by the nucleic acid molecule of the invention which only comprises naturally occurring amino acid residues, but it may also be a (poly) peptide containing modifications. These include covalent derivatives, such as aliphatic esters or amides of a carboxyl group, 0-acetyl derivatives of hydroxyl containing residues and N-acyl derivatives of amino group containing residues. Such derivatives can be prepared by linkage to reactable groups which are present in the side chains of amino acid residues and at the N-and C-terminus of the protein. Furthermore, the (poly) peptide can be radiolabeled or labeled with a detectable group, such as a covalently bound rare earth chelate, or conjugated to a fluorescent moiety. The (poly) peptide of the present invention can be, for example, the product of expression of a nucleotide sequence encoding such a (poly) peptide, a product of chemical modification or can be purified from natural sources, for example, viral preparations. Furthermore, it can be the product of covalent linkage of (poly) peptide domains.

The peptides/ (poly) peptides may also be produced by biochemical or synthetic techniques. Those methods are known to those of ordinary skill in the art (see, e. g. Merrifield, J. Am. Chem. Soc. 85 (1963), 2149-2146; Stewart,"Solid Phase Peptide Synthesis", WH Freeman Co, San Francisco (1969); Scopes, "Protein Purification", Springer Veriag, New York, Heidelberg, Berlin (1987) ; Janson,"Protein Purification, Principles, High Resolution Methods and Applications", VCH Publishers, New York, Weinheim, Cambridge (1989) ; Wrede,"Concepts in Protein Engineering and Design", Walter de Gruyter, Berlin, New York (1994)).

Additionally, within the scope of the invention are peptides/ (poly) peptides wherein the above mentioned amino acid (s) and/or peptide bonds have been replaced by functional analogs, inter alia by peptidomimetics. Peptidomimetics is well known in the art and corresponding art describing this method are mentioned below. Therefore, the present invention also encompasses functional derivatives and/or

analogues of said peptides comprising a specific peptide derived form the herein described splice-variants of FABP, in particular of H-FABP. Further methods for the preparation of peptides/ (poly) peptides are described in Sambrook et al., loc. cit. , or in Oxender and Fox (1987) "Protein Engineering", Alan Liss Inc. New York. Protein preparation of chemical derivates and/or analogues are described in, for example, Beilstein"Handbook of Organic Chemistry", Springer Edition New York, or in "Organic Synthesis", Wiley, New York.

The present invention also relates to a fusion protein comprising the inventive polypeptide of the invention or (a) fragment thereof.

Therefore, in addition to the inventive splice-variants of FABP of the present invention, said fusion protein can comprise at least one further domain, said domain being linked by covalent or non-covalent bonds. The linkage can be based on genetic fusion according to the methods known in the art (Sambrook et al., loc. cit. , Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N. Y. (1989) ) or can be performed by, e. g., chemical cross-linking as described in, e. g. , WO 94/04686. The additional domain present in the fusion protein comprising the (poly) peptide of the invention may preferably be linked by a flexible linker, advantageously a (poly) peptide linker, wherein said (poly) peptide linker preferably comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of said further domain and the N-terminal end of the peptide, (poly) peptide or antibody or vice versa. The above described fusion protein may further comprise a cleavable linker or cleavage site, which, for example, is specifically recognized and cleaved by proteinases or chemical agents.

Additionally, said at least one further domain may be of a redefined specificity or function. In this context, it is understood that the (poly) peptides of the invention may be further modified by conventional methods known in the art. This allows for the construction of fusion proteins comprising the (poly) peptide of the invention and other functional amino acid sequences, e. g., nuclear localization signals, transactivating domains, DNA-binding domains, hormone-binding domains, protein tags as also

described herein above. Like, e. g. GST, GFP, h-myc peptide, FLAG, HA peptide, Strep), The invention also relates to specific antibodies which specifically recognize the inventive polypeptide, i. e., the specific splice variants/isoforms of H-FABP. However it is also envisage that said antibodies do not only recognize the splice variants of human FABP but also homologous splice variants in other (mammalian) species.

The general methodology for producing antibodies is well-known and has, for monoclonal antibodies, been described in, for example, Köhler and Milstein, loc. cit and reviewed in J. G. R. Hurref, loc. cit. ). In accordance with the present invention the term"antibody"relates to monoclonal or polyclonal antibodies. Polyclonal antibodies (antiserum) can be obtained according to conventional protocols. Antibody fragments or derivatives have been described herein above and comprise F (ab') 2, Fab, Fv or scFv fragments; see, for example, Harlow and Lane,"Antibodies, A Laboratory Manual", CSH Press 1988, Cold Spring Harbor, NY. Preferably the antibody of the invention is a monoclonal antibody. Furthermore, in accordance with the present invention, the derivatives of the invention can be produced by peptidomimetics. In the context of the present invention, the term"aptamer"comprises nucleic acids such as RNA, ssDNA (ss = single stranded), modified RNA, modified ssDNA or PNAs which bind a plurality of target sequences having a high specificity and affinity. Aptamers are well known in the art and, inter alia, described in Famulok, Curr. Op. Chem. Biol.

2 (1998), 320-327. The preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sites (Gold, loc. cit). Said other receptors may, for example, be derived from said antibody etc. by peptidomimetics. The specificity of the recognition implies that other known proteins, molecules are not bound. A suitable host for assessing the specificity would imply contacting the above recited compound comprising an epitope of the nucleic acid"mblecule or the (poly) peptide of the invention as well as corresponding compounds e. g. from protein or nucleic acid molecules known in the art, for example in an ELISA format and identifying those antibodies etc. that only bind to the compound of the invention but do not or to no significant extent cross-react with said corresponding compounds.

The invention also provides for compositions comprising the inventive polynucleotide, the polypeptide or the antibody described herein above. Most preferably said composition is a pharmaceutical or a diagnostic composition to be used and employed in the diagnosis or treatment of coronary diseases, like CHF, or the induction of an"oxygen sparing effect". Said"oxygen sparing effect"is a result of the different oxidation states of the carbon atoms of carbohydrates and fatty acids. The complete oxidation process of glucose is accompanied by less consumption of oxygen than fatty acid oxidation. In particular, in line with the invention, an overexpression of splicevariants without the ability to bind to fatty acids results in an enhancement of the metabolism of glucose and therefore in an induction of the "oxygen sparing effect". Therefore said effect is relevant in the therapy of hypertrophic cardiomyopathy (HCM) and hypertrophic obstructive cardiomyopathy (HOCM).

The figures show: Fig. 1: Fluorescence of cardiomyocytes that were stimulated by indicated molecules, infected with a recombinant adenovirus (AV 151, Flag-CFP) and analysed 48 hrs later. PE: Phenylephrine (100 uM) ; LIF : Leukemia inhibitory factor (1 nglml) ; ET-1: Endothelin-1 (10 nM) ; ISO : Isoprenaline (10 pM).

Fig. 2: Fluorescence of cardiomyocytes that were stimulated by indicated molecules, infected with a recombinant adenovirus (AV 199, Flag-H-FABP-CFP) and analysed 48 hrs later. PE: Phenylephrine (100 uM) ; LIF : Leukemia inhibitory factor (1 ng/ml) ; ET-1 : Endothelin-1 (10 nM) ; ISO : Isoprenaline (10 pM).

Fig. 3: Graphical evaluation of a Fluorescence Hypertrophy Assay. Primary cardiomyocytes from neonatal rats were stimulated/not stimulated with LIF and infected with a recombinant adenovirus for the recombinant expression of GFP (AV44) or GFP and HA-H-FABP (AV138). After 48 hrs fluorescence intensity was determined and quantified. LlF : Leukemia inhibitory factor (1 ng/ml) Fig. 4: Graphical evaluation of immunoblot analyses of stimulated cardiomyocytes.

Relative expression of phosphorylated forms of Stat3, p42 Erk, p44 Erk or

PKCp (detected using anti-phospho-specific antibodies used at dilutions of 1: 1000, from NEB) or total protein expression as for DCMAG-1 or MHC (own antibody and from Sigma respectively, used at 1: 1000 dilution) in cells expressing either control virus (AV151) or FABP virus (AV199) ; n=4.

Fig. 5: Graphical evaluation of a PKB activity assay of stimulated cardiomyocytes based on an immunoblot analysis using a phosphorylation dependent antibody. FABP expression (column 2) resulted in a 2.6 fold increase in PKB kinase activity compared to cells expressing control virus only (column 1); n=5.

The invention is now described by reference to the following biological examples which are merely illustrative and are not to be construed as a limitation of scope of the present invention.

Exampie I : Isolation of cardiomyocytes and corresponding infection protocol Primary cardiomyocytes from neonatal rats were isolated and cultivated in the presence of different hypertrophy inducing agents. The cells were infected with recombinant adenoviruses to express the green-fluorescent-protein. Two days later signal intensity for each stimulation was measured by fluorescence microscopy in combination with a digital camera.

Example If : Isolation of primary cardiomyocytes from neonatal rats Neonatal rats (P2-P7) were sacrificed by cervical dislocation. The ventricles of the beating hearts were removed and cardiomyocytes were isolated with the"Neonatal Cardiomyocyte Isolation System" (Worthington Biochemicals Corporation, Lakewood, New Jersey) according to the protocol. Briefly, the ventricles were washed twice with ice cold Hank's Balanced Salt Solution without Potassium and Magnesium (CMF- HBBS) and minced with a scalpel to an average volume of one cubic millimeter. The heart tissue was further digested over night with trypsin at 10°C. Next morning trypsin inhibitor and collagenase were added. After an incubation at 37°C and mild agitation for 45 minutes the cells were dispersed by pipetting. The solution was further purified

by 70 u. m mesh (Cell Strainer) and centrifuged twice for 5 minutes at 60 x g. The cell pellet was resuspended in plating medium and counted. Cells were seeded with a density of 2.5 x 105/cm2 on gelatine (Sigma, Deisenhofen) coated dishes. The next morning cells were washed twice with DMEM and maintenance medium was added.

Plating medium: DMEM/M-199 (4/1); 10% Horse serum, 5% Fetal calf serum; 1 mM Sodiumpyruvate; Antibiotics and antimycotics Maintenance medium: DMEM/M-199 (4/1) ; 1 mM Sodiumpyruvate Example III : Stimulation of isolated cardiomyocytes from neonatal rats Stimulation of primary cardiomyocytes from neonatal rats (pCMs) was started two to six hours after medium was changed to maintenance medium. Different stimuli or combinations of stimuli were added to the cells. Used stimuli were: Phenyiephrine (PE) at 100 uM (Sigma) Leukemia inhibitory factor (LIF) at 1 nglml (Roche Diagnostics) Endothelin-1 (ET-1) at 10 nM (Roche Diagnostics) Isoprenaline (ISO) an 10 pM (Sigma) Directly after stimulation pCMs were infected with recombinant adenoviruses at a MOI of five. Cells were incubated for 48 hours at humidified atmosphere at 37°C and 5% C02 followed by a measurement of the fluorescence intensity.

Example IV : Generation of recombinant adenoviruses Recombinant adenoviruses were produced according to the simplified system developed by He et al. (He TC, Zhou S, da Costa LT, Yu J, Inter KW and Vogelstein B (1998) : A simplified system for generating recombinant adenoviruses.

Proc. Nati. Acad. Sci. USA. 95: 2509-2514). To generate a recombinant adenovirus genome in order to express the green-fluorescent-protein (GFP), the pAdTrack vector was combined with the pAdEasy-1 plasmid by homologous recombination.

Briefly, 5 pg of the pAdTrack plasmid were linearized with the restriction enzyme Pme I and gel-purified. Approximately 100 ng of the linearized vector were combined with 100 ng of the pAdEasy-1 plasmid and aqua bidest. was added to a final volume

of 7 pi. This solution was combined with 20 pi of electro-competent bacteria (BJ5183) and transferred to an electroporation cuvette (2.0 mm). The electroporation was performed using the Bio-Rad Gene Pulser (2.500 V, 200 Ohms, 25 uFD). Then 500 pi LB-medium were added. The bacterial culture was incubated at 37°C for 20 minutes in a bacterial shaker and afterwards plated on two LB-agar plates (1/10, 9/10) containing 50 ug/ml Kanamycin. After overnight incubation at 37°C tweive of the smallest colonies were picked and grown for at least 12 hours in 2 ml LB-medium (50 jjg Kanamycin) at 37°C in a bacterial shaker. Plasmid DNA from these overnight cultures was purified by alkaline lysis and digested with the restriction enzyme Pac I.

One of the positive clones which showed two fragments after cleavage (30 kb and 3.0 or 4.5 kb) was transformed into the bacterial strain DH5a by electroporation (2.500 V, 200 Ohms, 25 uFD). A single colony was picked and transferred into 500 mi LB-medium (50 pg/ml Kanamycin) and grown for 12 to 16 hours in a bacterial shaker. Plasmid DNA was prepared by the Tip-500 column (Qiagen, Hilden, Germany) according to the manufacturers instructions. Then 10 lug DNA were digested with Pac I, ethanol precipitated and resuspended in 40 pi H20 (cell culture grade).

The packaging was performed in HEK 293 cells (ATCC CRL-1573) by lipofection.

The day before transfection cells were seeded into two T-25 flask (2 x 106 cells each) in DMEM (10% fetal calf serum). For each flask 20 pi of the Pac I digested adenovirus genome was mixed with 20 pi of Lipofectamine (GIBCO BRL) in 500 pl OptiMem I medium and incubated for 15 minutes at room temperature. Meanwhile cells were washed twice with 4 mi serum-free DMEM. Then 2.5 ml OptiMem I was added to each flask followed by the DNA Lipofectamine solution. After incubation for 6 hrs at 37°C and 5% C02 medium was changed to DMEM (10% fetal calf serum).

Packaging was monitored by GFP expression of transfected cells. Cells were harvested after 7 to 14 days, depending on the efficiency of the packaging.

To harvest the cells they were detached by pipetting. Cells were sedimented by centrifugation at 100 x g for 5 minutes and the pellet was resuspended in 1 ml of (20 mM Tris pH 8.0, 2 mM MgCI2, 140 mM NaCI, 3 mM KCI). After three freeze-thaw cycles in liquid nitrogen and a 37°C waterbath the cell lysate was centrifuged at 150 x g. For the amplification of the recombinant adenovirus 80% of the supernatant were

used. The rest was stored at-80°C after adding glycerol to a final concentration of 25%.

The first amplification was performed in one T-25 flask with HEK 293 cells at a density of 70 to 80%. Cells were harvested and lysed as described above. The virus titer was determined after two to four rounds of further amplification in T-75 flasks.

The titer of infectious particles was determined by end-point-dilution with HEK 293 cells based on the TCID 50 method (Mahy and Kangro, Virology Methods Manual, Academic Press. p37).

An adenovirus to recombinantly express H-FABP and GFP bicistronicaily was generated as described above. Briefly, the pAdTrack-CMV. vector was linearized with EcoR I, gel purified and incubated with T4-polymerase and desoxynucieotide- triphophates (dNTPs) to get blunt ends. The vector was related, amplified and cut with Not I and Xba i. Then an adaptor (Not I-Xba I-EcoR I-Pme I-Xho I-STOP-HindIII- Nhe I) was inserted. Next the sequence for the Influenza Virus Hemagglutinine- epitope (Xba t-ATG TAC CCA TAC GAT GTA CCT GAC TAT GCG GGC TAT CCC TAT GAC GTC CCG GAC TAT GCA GGA TAT CCG TAT GAC GTT CCA GAT TAC GCG GGA TCC CCC GGG CTG CAG-EcoR l) (SEQ ID NO: 10) was inserted into the Xba I and EcoR I restriction sites. The final vector was cut with EcoR I and Xho I and ligated to a H-FABP PCR fragment comprising the whole open reading frame flanked by an EcoR I site on the 5'and a Xho I site at the 3'end. Both sites were added in frame by PCR primers. A human heart cDNA library was used as a template for the standard PCR. The resulting vector was linearized with Pme I and recombinant adenoviruses were generated as described in example 3.

Another series of adenoviruses for the monocistronic expression of recombinant proteins were generated based on the pShuttle vector In the first step the pShuttle plasmid was cut with Sal I and Kpn I, gel purified, blunted with T4-polymerase and related to eliminate the multiple cloning site. The vector was digested with EcoR I,

blunted with T4-polymerase and religated to get rid of the single EcoR I site in the vector backbone. Next the vector was linearized with Bgl II and desphosphorylated. A whole expression cassette for a Flag-CFP (cyano variant of GFP) fusion protein derived from a modified pCI (Promega) vector was inserted into the Bgl II site. The new construct was named plasmid &num 151.

The pi vector (Promega) was modified in the following way. It was cut with BsrG I, blunted with Klenow-fragment in the presence of dNTPs and related to eliminate the BsrG I site. The new vector was cut with Nhe I and Not I and gel purified. A PCR fragment containing the coding region for the CFP and the following restriction sites was inserted into the Nhe 1 and Not I sites.

Spe I-Xba I-EcoR I-Xho I-CFP coding region (2. codon until end)-STOP-Not I The pECFP-C1 plasmid (Clontech) served as a template for the PCR amplification. The new vector was cut with Xba I and EcoR I and gel purified. The coding region for the Flag epitope was constructed by oligo annealing and inserted into the Xba I and EcoR I sites.

Flag oligo 5' : CTA GAT CCA CCA TGG ATT ACA AGG ATG ACG ACG ATA AGG (SEQ ID NO : 11) Flag oligo 3' : AAT TCC TTA TCG TCG TCA TCC TTG TAA TCC ATG GTG GAT (SEQ ID NO : 12) (the first ATG of the coding region is indicated in bold) Next the whole expression cassette was isolated by digestion of this vector with BgI II and BamH I and gel purification of the smaller band. The resulting fragment was inserted into the Bgi il site of the vector described above to get plasmid #151. Plasmid #151 was linearized with Pme I and gel purified. The resulting recombinant adenovirus AV 151 for the expression of a Flag-CFP fusion protein was generated as described in example 3.

To get the recombinant adenovirus AV 199, which led to the expression of a Flag-H- FABP-CFP fusion protein, plasmid #151 was cut with EcoR I and Xho I and gel

purified. A PCR fragment containing the entire coding region of H-FABP without stop codon was inserted into the EcoR I and Xho I sites. To do this the EcoR I site was added in frame to the 5'end of the coding region and the Xho I site in frame to the 3' end by the PCR primers. Again a human heart cDNA library served as template for the PCR amplification. The resulting plasmid was cut with Pme I and gel purified.

Recombinant adenoviruses were generated as described in example 3.

Example V : Morphometric analysis of stimulated cardiomyocytes Morphometric analyses of cardiomyocytes were performed 48 hrs after stimulation.

The PE stimulation led to larger cells with increased in width whereas the LIF stimulation resulted in an elongation of pCMs. The ET-1/PE stimulation gave similar results as the PE stimulation, while ET-1 stimulated cells were only slightly larger than unstimulated cells. The ET-1/ISO/LIF stimulation in comparison with LIF stimulated cells in contrast led to a slight increase in width and length.

The same pattern of morphological alteration was seen in control virus infected cells after stimulation (Fig. 1). In particular the elongation of LIF stimulated cells was very characteristic.

The recombinant over expression of a Flag-H-FABP-CFP fusion protein in FABP virus infected cells on the other hand led to a profound hypertrophy of pCMs in unstimulated and under all tested stimulation conditions (Figure 2). The most prominent effect was observed after LIF stimulation.

Example VI : Fluorescence analysis of GFP expressing cardiomyocytes Determination of fluorescence intensity of GFP expressing pCMs was performed 48 hours after stimulation and infection. Cells were analysed under the fluorescence microscope (Axiovert 100S, Zeiss, Jena, Germany), images were captured with a digital camera (LAS-1000, Fuji) and analysed with the AIDA software package (Raytest). For each well of a 6-well-plate, which represents one individual stimulation experiment, five randomly selected regions were analysed with the 20x objective and a GFP filter set (AF-Analysetechnik, Tubingen, Germany). Time of exposure was chosen to the maximal value before saturation. The five individual values were used

to calculate a mean value for each stimulation. After subtraction of background (signal of uninfected cells) the mean values for nine independent experiments were calculated and analyzed. The comparison of the fluorescence intensities between unstimulated and PE or LIF stimulated cells showed a clear increase for the latter two. The ET-1/PE stimulation led to a similar value, while ET-1/ISO/LIF stimulated cells showed a further increase in the GFP signal. ET-1 stimulation on the other hand resulted in an intensity which was marginally above background. These observations augmented the assumption that there was a good correlation between hypertrophy status and fluorescence intensity of stimulated pCMs.

Fig. 3 shows the different fluorescence intensities of unstimulated and LIF stimulated cells either infected with a control adenovirus (GFP virus-AV 44) or with a H-FABP expressing adenovirus (GFP and HA-H-FABP-AV 138), There was again a good correlation between the increased hypertrophy of LIF stimulated and unstimulated cells measurable. In addition there was a clear hypertrophic effect of recombinant H- FABP expression observable.

Example Vil : lmmunoblot analysis of stimulated cardiomyocytes Primary cardiomyocytes were analysed for the expression or activation of a number of marker proteins that have been identified in the company as up-regulated upon onset of hypertrophy. The effect of FABP expression on these marker proteins was tested by immunoblotting for protein expression or for amount of the phosphorylated (and thus activated) protein in question. The tested proteins were P-Stat3, P-p42 Erk, P-p44 Erk, DCMAG-1, MHC, P-PKCß.

Primary cardiomyocytes were cultured as described above and either control virus or Flag-FABP-CFP virus expressed. After 48h of cultivation in 6 well plates seeded at a density of 5x1 Oe5 cells/well, ceils were washed once in PBS and then lysed in 100, ui loading dye (50% glycerol, 10% SDS, 500mM DTT, 0.5M Tris buffer pH 6.8 ; 1: 3 diluted with lysis buffer : 1% Triton X-100, 200mM Tris pH 7. 4, 100mM NaF, 1 Boehringer Mini Tablet, 1mM PMSF). Cells were scraped into the loading dye, transferred to an Eppendorf tube, vortexed for 1 min, boiled for 5 min and then centrifuged for 1 min at 15,000 rpm in an Eppendorf centrifuge. 40p1 of the resulting

mixture were loaded onto a 10% SDS-polyacrylamid gel and separated at 160V for 1 hour. Samples were transferred onto nitrocellulose (Hybond, Amersham) at 75V for 1 hr in standard transfer buffer (Sambrook et al. (1989). Molecular Cloning : a laboratory manual, 2. Edition, Cold Spring Harbor, NY).

Proteins were detected by Western Blotting as follows : Nitrocellulose filters were blocked for 45 min in TBS-0. 1 % Tween, 1 % BSA, then incubated with the appropriate antibody at 1: 1000 dilution in TBS-0. 1% Tween, 1% BSA for a minimum of 2 hrs (anti-phospho-specific antibodies for Stat3, p42 Erk, p44 Erk and PKCp from NEB, polyclonal anti-DCMAG-1 by MediGene, anti-MHC form Sigma).

Membranes were washed 4 times in T8S-0. 1% Tween, and incubated for 1 hr in TBS-0. 1 % Tween, 1 % BSA with secondary anti-mouse or anti-rabbit IgG coupled to HRP (1: 10.000). Protein bands were visualized using Amersham ECL Plus kit according to manufacturers instructions and bands were detected and quantified using a Fuji LAS1000 digital camera. As a loading control blots were stripped (10% SDS, 100mM Tris pH 6.8, 1% ß-mercaptoethanol at 50°C for 30 min) and reblotted by above method for expression of virus by detecting the Flag-tagged construct using the M2 anti-Flag antibody from Sigma (1: 1000). Protein expression was calculated as fold over expression detected in control virus expressing cells.

Fig. 4 shows that the over-expression of FABP lead to an up-regulation of known markers for hypertrophy such as MHC or DCMAG-1 and to an activation of PKCb (as P-PKCß is up-regulated), whereas other tested proteins as P-Stat3, P-p42 Erk or P- p44 Erk were not affected.

In addition it was found that expression of FABP activates Protein kinase B (PKB/Akt) as could be measured in a PKB kinase activity assay. The assay was done using kit no 9, 840 from NEB. Briefly cells were lysed and PKB precipitated from the cleared lysate using anti-PKB antibody. The precipitates were washed and incubated with GSK3p fusion protein as a substrate as well as ATP. The mixture was incubated for 30min at 30°C and stopped by the addition of loading dye. The mixture was separated using a 12.5% SDS polyacrylamid get and transferred as per standard protocol. Phosphorylated GSK3ß fusion protein was detected by Western Blotting using an anti-phospho site-specific antibody, anti-mouse HRP and LumiGlo from

NEB. Signals were detected and quantified using the LAS1000 digital camera and AIDA software.

Fig. 5 shows that the expression of H-FABP leads to a 2.6 fold increase in PKB kinase activity compared to cells expressing control virus only.

These results suggest that markers that are associated with the development of the hypertrophic phenotype (e. g. PKC beta, DCMAG, MHC, PKB) are increased upon expression of H-FABP in primary neonatal cardiomyocytes. In addition the subset of markers activated taken together with the particular phenotype exhibited by H-FABP expressing pCMs suggest that overexpression of FABP results in the development of adaptive hypertrophy.

In conclusion, the detailed analysis of the gene expression pattern in tissue samples from normal and diseased human hearts revealed a marked increase in genes, which are involved in oxidation of free fatty acids (FFAs). This observation sets the basis for a new therapeutic intervention (the reduction of fatty acid consumption) in congestive heart failure.

The product of one of the affected genes-H-FABP-showed a completely new effect after overexpression in primary cardiomyocytes from neonatal rats (pCMs). It induced a remarkable hypertrophy with a parallel organization of sarcomeres, which is usually found in cardiomyocytes derived from concentric hypertrophied hearts. This new function of H-FABP sets the basis for an additional therapeutic intervention, in particular the link between H-FABP and signaling molecules like PKCß and PKB, as the induction of a parallel sarcomere organization could be the prerequisite for the transition to the maladative/insufficiency state of the heart. Also the fatty acid binding function of H-FABP could be a target for the therapy of heart failure as it is known to correlate with the amount of FFA 9-oxidation of the heart.

Example Vlil : Characterization of novel H-FABP variants As mentioned herein above, originally, the H-FABP gene was described to contain 4 exons (Genbank entry: U57623, cDNA). Briefly, by combining EST-clustering and RT-

PCR, we discovered a coding region of 67 nts that was previously thought to be exclusively intronic. Thus, the entire gene contains 5 exons. Furthermore, it has been determined that three different transcripts are synthesized from the same gene due to alternative splicing. FABP3p, a novel splice variant, is produced by the usage of an alternative 3'-splice site in exon 2A This results in a frame shift and the stop codon is now located in exon 3. Exon 2A can also be included completely in the mature transcript. This results in the formation of FABP3y, a further splice variant; the stop- codon is in exon 2A. Therefore, it has surprisingly found that three isoforms of H-FABP in healthy heart tissue as well as in DCM patient exist.

In detail, the experiments have been carried out as follows : The FABP3a sequence SEQ ID NO: 1 was analysed for possible isoforms using EST clustering and assembly tools (Compugen). With this procedure, an EST (Al367747, representing an incomplete version of FABP3p) was identified that contained an insertion compared to FABP3a. Analysis of the genomic sequence of FABP3 indicated the existence of a possible splice site in this region of interest.

The existence of an isoform containing the insertion (SEQ ID NO: 7) was confirmed by RT-PCR from human adult heart RNA using the primers FABPfw3 (recognizing specifically the novel sequence (SEQ ! D NO: 7) as a part of the nove ! exon 2a of FABP3p) and FABPrev2 (hybridizing to a region in exon 4). The fragment was cloned into TOPO pCR2. 1 (Invitrogen) and sequenced.

The region of interest was also examined by RT-PCR using the primers #23 (recognizing the 5'-end of the FABP3a coding region) and FABPrev3a (recognizing the novel sequence, SEQ ID NO: 7 of FABP3p. Cloning and sequencing of this fragment revealed the existence of sequence SEQ ID NO: 9 as part of the novel exon 2a, which is present in the novel isoform FABP3y. Full-length cloning of the isoforms FABP3ß (SEQ ID NO: 3) and FABP37 <400>5 occurred by RT-PCR from human adult heart RNA using the primer combination #23 and FABPb-attb2-end for FABP3p as well as with the primer combination #23 and FABPg-attb2 for FABP3y. Cloning occurred in pDONOR201 (Invitrogen). The clones were verified by sequencing.

The protein sequences of FABP3p and FABP3y are shown in SEQ ID NO: 4 and SEQ ID NO: 6, respectively. The partial nove ! protein sequences of FABP3) and FABP3y that differ from FABP3a are shown in SEQ ID NO: 8 and SEQ ID NO: 10.

Primers used: #23 : 5'-TACAAAAAAGCAGGCTctatggtggacgctttcctgggcacctggaagctagtgg-3 '

FABPfw3: 5'-ctccagctgggtgaccctgtgc-3' FABPrev3a: 5'-gcacagggtcacccagctggag-3' FABrev2: 5'-tttggccttggctctgctttattg-3' FABPb-attb2-end : 5'-gaaagctgggtgattagctcccgcacaagtgtggtctc-3' FABPg-attb2 : 5'-gaaagctgggtggaacatcctggctctgtgcttg-3'