WO2003006687A2

WO2003006687A2 - Novel target genes for diseases of the heart

Info

Publication number: WO2003006687A2
Application number: PCT/EP2002/007704
Authority: WO
Inventors: Birgit Reuner; Daniela Bunk; Thomas Henkel
Original assignee: Medigene AG
Current assignee: Medigene AG
Priority date: 2001-07-10
Filing date: 2002-07-10
Publication date: 2003-01-23
Anticipated expiration: 2004-01-10
Also published as: WO2003006687A3

Abstract

The present invention relates to a variety of genes abnormally expressed in heart tissue as well as to fragments of such genes. Assessment of the expression level of these genes may be used for testing the predisposition of mammals and preferably humans of a heart disease or for an acute state of such a disease. Preferred diseases in accordance with the invention are congestive heart failure, dilative cardiomyopathy, hypertrophic cardiomyopathy and ischemic cardiomyopathy. The present invention further relates to methods of identifying compounds capable of normalizing the expression level of the aforementioned genes and of further genes affected by the abnormal expression. The identified compounds may be used for formulating compositions, preferably pharmaceutical compositions for preventing or treating diseases. They may be used as lead compounds for the development of medicament having an inproved efficiency, a longer half-life, a decreased toxicity etc. and to be employed in the treatment of heart diseases. Included in the invention are also somatic gene therapy methods comparing the introduction of at least one functional copy of any of the above-mentioned genes into a suitable cell. Finally, the invention relates to non-human transgenic animals comprising at least one of the aformentioned genes in their germ line. The transgenic animals of the invention may be used for the development of medicaments for the treatment of heart diseases.

Description

Novel target genes for diseases of the heart

A variety of documents is cited throughout this specification. The disclosure content of said documents is herewith incorporated by reference.

The present invention is based on the finding that a variety of genes is abnormally expressed in diseased heart tissue. Assessment of the expression level of these genes may be used for testing the predisposition of mammals and preferably humans for a heart disease or for an acute state of such a disease. Diseases that preferably relate to the invention are congestive heart failure, dilative cardiomyopathy, hypertrophic cardiomyopathy and ischemic cardiomyopathy. The present invention further relates to methods of identifying compounds capable of normalizing the expression level of the aforementioned genes and of further genes affected by the abnormal expression. The identified compounds may be used for formulating compositions, preferably pharmaceutical compositions for preventing or treating diseases. They may also be used as lead compounds for the development of medicaments having an improved efficiency, a longer half-life, a decreased toxicity etc. and to be employed in the treatment of heart diseases. Included in the invention are also somatic gene therapy methods comprising the introduction of at least one functional copy of any of the above-mentioned genes into a suitable cell. Finally, the invention relates to non-human transgenic animals comprising at least one of the aforementioned genes in their germ line. The transgenic animals of the invention may be used for the development of medicaments for the treatment of heart diseases.

Referring to studies of the American Heart Association about 60 million people in the USA suffer from Cardiovascular diseases like high blood pressure (50.0 mio), Coronary heart disease (12.4 mio), Myocardial infarction (7.3 mio), Angina pectoris (6.4 mio), Stroke (4.5 mio), Congenital cardiovascular defects (1.0 mio), and Congestive heart failure (4.7 mio). Hence, it follows that 20 per cent of whole population is affected. The mortality was 949,619 in 1998 in the USA, which means that about 40 % of all deaths were caused by Cardiovascular diseases. Since 1900 Cardiovascular diseases are the number one cause of death (1918 was an exception) with one death every 33 seconds on average. At present there is no causal treatment for congestive heart failure available.

Accordingly, the technical problem underlying the present invention was to provide a new generation of tools useful in diagnosis, prognosis, prevention and treatment of heart-related diseases.

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

Thus, the present invention relates to a method for identifying a subject at risk for a disease of the heart, comprising the step of quantitating the amount of at least one RNA in the heart tissue of a subject, whereby (a) said at least one RNA encodes an amino acid sequence selected from the group consisting of: (aa) the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10;_(ab) an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (aa); (ac) the amino acid sequence of (aa) with at least one conservative amino acid substitution; (ad) an amino acid sequence that is an isoform of the amino acid sequence of any of (aa) to (ac); and (ae) an amino acid that is encoded by a DNA molecule the complementary strand of which hybridizes in 4xSSC, 0.1% SDS at 65°C to the DNA molecule encoding the amino acid sequence of (aa), (ac) or (ad); and/or (b) said at least one RNA is transcribed from the DNA sequence of SEQ ID NO: 11 , the DNA sequence of SEQ ID NO: 12, the DNA sequence of SEQ ID NO: 13, the DNA sequence of SEQ ID NO: 14, the DNA sequence of SEQ ID NO: 15, the DNA sequence of SEQ ID NO: 16, the DNA sequence of SEQ ID NO: 17, the DNA sequence of SEQ ID NO: 18, the DNA sequence of SEQ ID NO: 19, the DNA sequence of SEQ ID NO: 20, the DNA sequence of SEQ ID NO: 21 , the DNA sequence of SEQ ID NO: 22, the DNA sequence of SEQ ID NO: 23, the DNA sequence of SEQ ID NO: 24 or the DNA sequence as depicted in Figure 4F, the DNA sequence of SEQ ID NO: 25 or the DNA sequence as depicted in Figure 6B, the DNA sequence of SEQ ID NO: 26 or the DNA sequence as depicted in Figure 7B the DNA sequence of SEQ ID NO: 27 or the DNA sequence as depicted in Figure 8B or the DNA sequence of SEQ ID NO: 28 or the DNA sequence as depicted in Figure 2B or a degenerate variant thereof.

The term "disease of the heart" means, in accordance with the present invention, any disease that affects the normal function of the heart. This definition includes hereditary as well as acquired diseases such as diseases induced by a pathogen or diseases due to lack of exercise.

Several diseases of the heart are, for example, rheumatic fever/ rheumatic heart disease, hypertensive heart disease, hypertensive heart and renal disease, ischemic heart disease (coronary heart disease), diseases of pulmonary circulation (which include acute and chronic pulmonary heart disease), arrhythmias, congenital heart disease, angina and congestive heart failure.

The term "quantitating the amount of at least one RNA" is intended to mean the determination of the amount of mRNA in heart tissue as compared to a standard value such as an internal standard. The (internal) standard would advantageously be the amount of a corresponding RNA produced by a heart tissue not affected by a disease. Said (internal) standard would also include a mean value obtained from a variety of heart tissues not affected by a disease. A possible way to get samples of heart tissue is to take a biopsy (catheter) from the ventricular wall. Optionally, a standard would take into account the genetic background of the subject under investigation. Thus, quantitation of said subject's RNA is effected in comparison to the amount of RNA of one or a variety of samples of the same or a similar genetic background A variable number of "non-failing" humans (humans that do not show an indication for any heart disease) are compared with a variable number of patients that suffer a distinct heart disease like dilated cardiomyopathy. The determination can be effected by any known technology of analysing the amount of RNA produced in a sample such as a tissue sample. Techniques based on hybridisation like Northern-Blot, dot-blot, subtractive hybridisation, DNA-Chip analysis or techniques based on reverse transcription coupled to the polymerase chain reaction (RT-PCR) like differential display, suppression subtractive hybridisation (SSH), fluorescence differential display (FDD), serial analysis of gene expression (SAGE) or representational difference analysis (see e.g. Kozian, D.H., Kirschbaum, B.J.; Comparative gene-expression analysis. (1999) 17:73-77. Generally, it is preferred that the assay is performed as a high throughput assay. This holds also true for the further methods described herein and in accordance with this invention. Samples of RNA may be prepared as described in the appended examples.

The term "isoform" means a derivative of a gene resulting from alternative splicing, alternative polyadenylation, alternative promoter usage or RNA editing. Isoforms can be detected by

(a) in silico analysis (e.g. by clustering analysis of any types of expressed sequences or the corresponding proteins, by alignment of expressed sequences with chromosomal DNA, by interspecies comparisons or by analysis of the coding as well as non-coding sequences like promoters or regulatory RNA processing sites for SNPs or known mutations causing a disease).

(b) any type of hybridisation techniques (e.g. Northern blots, nuclease protection assays, microarrays) starting from RNA (as described in Higgins, S.J., Hames, D. RNA Processing: A practical approach Oxford University Press (1994), Vol. 1 and 2; Sambrook, Fritsch, Maniatis. Molecular Cloning, a laboratory manual. (1989) Cold Spring Harbor Laboratory Press).

(c) PCR-applications as well as hybridisation techniques starting from single strand or double strand cDNA obtained by reverse transcription, as described for example in Stoss, O., Stoilov, P., Hartmann, A.M., Nayler, O., Stamm, S. The in wVo minigene approach to analyse tissue-specific splicing. Brain Res. Brain Res. Protoc. (1999), 3:383-394.

Primers/probes for RT-PCR or hybridisation techniques are designed in a fashion that at least one of the primers/probes specifically recognizes one isoform. If differences in the molecular weight of isoforms are large enough to separate them by electrophoretic or chromatographic methods, it is also possible to detect multiple isoforms at once by employing primers/probes that flank the spliced regions. The isoforms are then sequenced and analysed as described in a).

The term "DNA molecule the complementary strand of which hybridizes in 4xSSC, 0.1 % SDS at 65°C to the DNA molecule encoding the amino acid sequence of (a), (c) or (d)" means that the two DNA molecules hybridize under these experimental conditions to each other. This term does not exclude that the two DNA sequences hybridize at higher stringency conditions such as 2xSSC, 0.1% SDS at 65°C nor does it exclude that lower stringency conditions such as 6xSSC, 0.1% SDS at 60°C allow a hybridization of the two DNA sequences.

Appropriate hybridization conditions for each sequence may be established on well-known parameters such as temperature, composition of the nucleic acid molecules, salt conditions etc.; see, for example, Sambrook et al., "Molecular Cloning, A Laboratory Manual"; CSH Press, Cold Spring Harbor, 1989 or Higgins and Hames (eds.), "Nucleic acid hybridization, a practical approach", IRL Press, Oxford 1985, see in particular the chapter "Hybridization Strategy" by Britten & Davidson, 3 to 15.

The term "degenerate variant" refers to the degeneracy of the genetic code. Said degenerarcy is know in the art and described in several text books, e.g. Lewin, Genes V, Oxford University Press 1994, Chapter 7. The invention is based upon the unexpected result that the certain genes coding for the protein sequences referred to above, preferred embodiments of which are given in examples 2 to 11 are deregulated in the comparison of one or more failing heart samples to one or more non-failing heart samples and lead to a downregulation of the described polypeptides measured by their respective mRNAs or cDNAs. The significant changes in gene expression levels suggest a causative role in congestive heart failure.

However such a causative role for one specific indication of the heart leads to the assumption that a deregulation of such gene(s) might play an important role in other diseases of the heart as well. Such involvement can easily be tested by methods well known in the art and described for example in example 1 of this invention by a comparison of the gene expression levels of such gene between a sample of a healthy mammal and of a mammal having the disease in question. Therefore the subject of this invention does not only relate to dilated cardiomyopathy but also to other diseases of the heart as specified throughout the specification.

It is well accepted in the art that upregulation of gene expression of a downregulated target gene by means of a gene therapeutic intervention, compensatory molecules or specific activators, for example of transcription or translation are potentially very promising therapeutic tools to treat a heart disease that is caused or promoted by the downregulation of such gene.

Further, in accordance with the present invention it has surprisingly been found that a variety of genes is aberrantly expressed in diseases associated with the heart and in particular in patients suffering from congestive heart failure. By performing the method of the invention which may be in vivo, in vitro or in silico, the diagnosis of a disease of the heart established by a different methodology may be corroborated. Alternatively, it may be assessed whether a subject that is preferably throughout this specification a human displaying no sign of being affected by a disease of the heart is at risk of developing such a disease. This is possible in cases where the aberrant expression of the gene defined herein above is causative of the disease or is a member of a protein cascade wherein another gene/protein than the one identified herein above is causative for said disease. In this regard, the term "causative" is not limited to mean that the aberrant expression of one gene as identified above or which is a member of said protein cascade is the sole cause for the onset of the disease. Whereas this option is also within the scope of the invention, expression the invention also encompasses embodiments wherein said aberrant is one of a variety of causative events that lead to the onset of the disease.

There is causal correlation between altered cellular function of cardiomyocytes and its protein composition. The latter is regulated by three main mechanisms: a. Gene expression b. Posttranscriptional modification (e.g. alternative splicing) c. Posttranslational modification

In a variation of the method of the invention quantitation of the above recited RNA is used to monitor the progress of a disease of the heart (said variation also applies to the method described herein below). This variation may be employed for assessing the efficacy of a medicament or to determine a time point when administration of a drug is no longer necessary or when the dose of a drug may be reduced and/or when the time interval between administrations of the medicament may be increased. This variation of the method of the invention may successfully be employed in cases where an aberrant expression of any of the aforementioned genes/genes as members of protein cascades is causative of the disease. It is also useful in cases where the aberrant expression of the gene/genes is the direct or indirect result of said disease.

When assessing the risk or the status of the disease, one or more of the RNA levels may be determined. Generally, the assessment of more than 1 , such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 different RNAs is expected to enhance the fidelity of the prognosis/diagnosis. However, the gain in fidelity would, as a rule, have to be weighted against the costs generated by such additional tests. Accordingly, it is preferred that one or two different RNA levels are determined for a first assessment. If deemed necessary or appropriate, further RNA levels may be determined.

The DNA sequence of SEQ ID NO: 24 or the DNA sequence as depicted in Figure 4F or a degenerate variant thereof recited in the methods of the invention described herein comprise the sequence of SEQ ID NO: 17, depicted in Figure 4B.

In a preferred embodiment of the method of the invention the amount of the said RNA is quantitated using a nucleic acid probe which is a nucleic acid comprising a sequence selected from the group consisting of:

(a) the DNA sequence of SEQ ID NO: 11, the DNA sequence of SEQ ID NO: 12 the DNA sequence of SEQ ID NO: 13, the DNA sequence of SEQ ID NO: 14 the DNA sequence of SEQ ID NO: 15, the DNA sequence of SEQ ID NO: 16 the DNA sequence of SEQ ID NO: 17, the DNA sequence of SEQ ID NO: 18 the DNA sequence of SEQ ID NO: 19, the DNA sequence of SEQ ID NO: 20 the DNA sequence of SEQ ID NO: 21 , the DNA sequence of SEQ ID NO: 22 the DNA sequence of SEQ ID NO: 23, the DNA sequence of SEQ ID NO: 24 or the DNA sequence as depicted in Figure 4F, the DNA sequence of SEQ ID NO 25 or the DNA sequence as depicted in Figure 6B, the DNA sequence of SEQ ID NO: 26 or the DNA sequence as depicted in Figure 7B the DNA sequence of SEQ ID NO: 27 or the DNA sequence as depicted in Figure 8B or the DNA sequence of SEQ ID NO: 28 or the DNA sequence as depicted in Figure 2B or a degenerate variant thereof

(b) a DNA sequence at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the DNA sequence of (a);

(c) a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; each of said amino acid sequences having at least one conservative amino acid substitution;

(d) a nucleic acid sequence that encodes an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (c);

(e) a nucleic acid sequence that encodes the amino acid sequence of (c) or (d) with at least one conservative amino acid substitution;

(f) a nucleic acid sequence that hybridizes in 4xSSC, 0.1% SDS at 65°C to the complementary strand of the DNA molecule encoding the amino acid sequence of (c), (d) or (e);

(g) a fragment of at least 15 nucleotides in length of (a) to (f); and

(h) a nucleic acid probe comprising a sequence that specifically hybridizes under physiological conditions to the nucleotide sequence selected from the group consisting of: (i) the DNA sequence of the RNA transcribed from the DNA sequence of SEQ ID NO 11 , the DNA sequence of SEQ ID NO: 12 the DNA sequence of SEQ ID NO 13, the DNA sequence of SEQ ID NO: 14 the DNA sequence of SEQ ID NO 15, the DNA sequence of SEQ ID NO: 16, the DNA sequence of SEQ ID NO 17, the DNA sequence of SEQ ID NO: 18, the DNA sequence of SEQ ID NO 19, the^" DNA sequence of SEQ ID NO: 20, the DNA sequence of SEQ ID NO 21 , the DNA sequence of SEQ ID NO: 22, the DNA sequence of SEQ ID NO: 23, the DNA sequence of SEQ ID NO: 24 or the DNA sequence as depicted in Figure 4F, the DNA sequence of SEQ ID NO: 25 or the DNA sequence as depicted in Figure 6B, the DNA sequence of SEQ ID NO: 26 or the DNA sequence as depicted in Figure 7B the DNA sequence of SEQ ID NO: 27 or the DNA sequence as depicted in Figure 8B or the DNA sequence of SEQ ID NO: 28 or the DNA sequence as depicted in Figure 2B; (ii) a DNA sequence at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the DNA sequence of (i); (iii) a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10 with at least one conservative amino acid substitution; (iv) a nucleic acid sequence that encodes an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (iii); (v) a nucleic acid sequence that encodes the amino acid sequence of (iii) with at least one conservative amino acid substitution; (vi) a nucleic acid sequence that hybridizes in 2xSSC, 0.1 % SDS at 65°C to the DNA molecule encoding the amino acid sequence of (iii), (iv) or (v), and a fragment of at least 15 nucleotides in length of (i) to (vi).

Advantageously, the nucleic acid sequence described herein above as well as the fragments thereof is/are preferably a DNA sequence(s) and is/are, preferably, detectably labeled. Appropriate labels include radioactive labels, wherein the radioactivity conferring molecules may be, e.g., ³²P, ³⁵S or ³H. Appropriate labels further include fluorescent, phosphorescent or bioluminescent labels or nucleic acid sequences coupled to biotin or streptavidin in order to detect them via anti- biotin or anti-streptavidin antibodies. Whereas any of the above mentioned probes specifically hybridizing to the aforementioned RNAs may be employed, it is preferred that fragments of the full length coding sequence such as oligomers of a length between 15 and 25 nucleotides are used. Examples of such oligomers are oligomers of 18, 21 or 24 nucleotides. Alternatively, the double strand formed after hybridization can be detected by anti-double strand DNA specific antibodies or aptamers etc.

In this regard, it is understood that the probe of SEQ ID NO: 13 and the mentioned variants thereof are used for quantitating the RNA of SEQ ID NO: 1 , but not to any of the other mentioned RNAs. In the following, appropriate pairs of RNAs and corresponding probes for assessing risks etc. of diseases of the heart are mentioned with the understanding that (i) appropriate variants of the probes as mentioned above may be used and (ii) said probes are specific for the corresponding RNA only but not for any of the other mentioned RNAs. These pairs are: SEQ ID NO: 2/SEQ ID NO: 14; SEQ ID NO: 3/SEQ ID NO: 15; SEQ ID NO: 4/SEQ ID NO: 16; SEQ ID NO: 5/SEQ ID NO: 17; SEQ ID NO: 6/SEQ ID NO: 18; SEQ ID NO: 7/SEQ ID NO: 19; SEQ ID NO: 8/SEQ ID NO: 20, SEQ ID NO: 9/SEQ ID NO: 21 and SEQ ID NO: 10/SEQ ID NO: 22. An appropriate pair of RNA and corresponding probe in the above described context is SEQ ID NO:7 and SEQ ID NO 25.

After hybridization, appropriate washing steps are performed in order to remove unspecific signals. Appropriate washing conditions include 2 washing steps at 65°C with 2xSSC, 0,1% SDS for 30 min (50 ml) and finally two washing steps with 50 ml of a solution containing O.lxSSC, 0.1% SDS for 30 min.; see also Sambrook et al., loc. cit, Higgins and Hames, loc. cit. After washing, the label is detected, depending on its nature. For example, a radioactive label may be detected by exposure to an X-ray film or by a phosphorimager. Alternatively, biotinylated probes can be detected by fluorescence, e.g. by using SAPE (streptavidin- phycoerythrin) with subsequent detection of the signal by a laser scanner.

In a further preferred embodiment, the invention provides a kit for identifying a subject at risk for a diseases of the heart or for monitoring the status or progression of a subject with a disease of the heart, comprising a means for detecting at least one RNA in a sample and means for detecting the level of RNA in the sample. In a preferred embodiment, the kit comprises at least one nucleic acid probe as described above and instructions for hybridizing of the nucleic acid probe with the nucleic acid molecules in the sample. In a preferred embodiment, the probe is detectably labeled, as discussed above. In another preferred embodiment, the nucleic acid probe is bound to a solid substrate. In addition, the invention relates to a method for identifying a subject at risk for a disease of the heart, comprising the step of quantitating the amount of a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, in the heart tissue or the serum of the blood of the subject. Further included are polypeptides encoded by any of the above recited nucleic acid sequences. This holds also true for any of the other embodiments in which the aforementioned polypeptides are employed.

This embodiment of the invention makes use of the option that detection may not only be at the level of the mRNA but also at the level of the polypeptide translated from the mRNA. Whereas it is not excluded that the level of mRNA strictly correlates with the level of polypeptide translated from the mRNA, this may not always be the case. Accordingly, it may be assessed whether the mRNA or the protein level, if different, is more appropriate to establish if the heart of a subject is prone to develop a disease of the heart. Factors that contribute to differences in the expression levels of mRNA and protein are well-known in the art and include differential mRNA-export to the protein-synthesis machinery as well as differences in the translation efficacy of different mRNA species. Other considerations influencing the choice of the detection level (in RNA or protein) include the availability of an appropriate screening tool, instrumentation of the lab, experience of the lab personnel and others. In a preferred embodiment of the method of the invention, the amount of the said polypeptide is quantitated using an antibody or an antigen-binding portion of said antibody that specifically binds a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution.

The antibody used in accordance with the invention may be a monoclonal or a polyclonal antibody (see Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, USA, 1988) or a derivative of said antibody which retains or essentially retains its binding specificity. Whereas particularly preferred embodiments of said derivatives are specified further herein below, other preferred derivatives of such antibodies are chimeric antibodies comprising, for example, a mouse or rat variable region and a human constant region. The term "specifically binds" in connection with the antibody used in accordance with the present invention means that the antibody etc. does not or essentially does not cross-react with (poly)peptides of similar structures. Cross-reactivity of a panel of antibodies etc. under investigation may be tested, for example, by assessing binding of said panel of antibodies etc. under conventional conditions (see, e.g., Harlow and Lane, loc. cit.) to the polypeptide of interest as well as to a number of more or less (structurally and/or functionally) closely related polypeptides. Only those antibodies that bind to the polypeptide of interest but do not or do not essentially bind to any of the other (poly)peptides which are preferably expressed by the same tissue as the polypeptide of interest, i.e. heart, are considered specific for the polypeptide of interest and selected for further studies in accordance with the method of the invention.

In a particularly preferred embodiment of the method of the invention, said antibody or antibody binding portion is or is derived from a human antibody or a humanized antibody.

The term "humanized antibody" means, in accordance with the present invention, an antibody of non-human origin, where at least one complementarity determining region (CDR) in the variable regions such as the CDR3 and preferably all 6 CDRs have been replaced by CDRs of an antibody of human origin having a desired specificity. Optionally, the non-human constant region(s) of the antibody has/have been replaced by (a) constant region(s) of a human antibody. Methods for the production of humanized antibodies are described in, e.g., EP-A1 0 239 400 and WO90/07861.

The specifically binding antibody etc. may be detected by using, for example, a labeled secondary antibody specifically recognizing the constant region of the first antibody. However, in a further particularly preferred embodiment of the method of the invention, the antibody, the binding portion or derivative thereof itself is detectably labeled.

Detectable labels include a variety of established labels such as radioactive (¹²⁵I, for example) or fluorescent labels (see, e.g. Harlow and Lane, loc. cit.). Binding may be detected after removing unspecific labels by appropriate washing conditions (see, e.g. Harlow and Lane, loc. cit.).

In an additionally preferred embodiment of the method of the invention, said derivative of said antibody is an scFv fragment.

The term "scFv fragment" (single-chain Fv fragment) is well understood in the art and preferred due to its small size and the possibility to recombinantly produce such fragments.

In a further preferred embodiment, the invention provides a kit for identifying a subject at risk for a diseases of the heart or for monitoring the status or progression of a subject with a disease of the heart, comprising a means for detecting at least one polypeptide as described above in a sample and means for detecting the level of the polypeptide in the sample. In a preferred embodiment, the kit comprises at least one antibody, binding portion thereof, or derivative thereof that specifically binds to the polypeptide, as described above and instructions for binding the antibody, binding portion thereof, or derivative thereof with the polypeptide in the sample. In a preferred embodiment, the antibody, binding portion thereof, or derivative thereof is detectably labeled. In another preferred embodiment, the kit provides another reagent that specifically binds the antibody, binding portion thereof, or derivative thereof and that is detectably labeled, as discussed above. In another preferred embodiment, the antibody, binding portion thereof, or derivative thereof is bound to solid substrate.

In a preferred embodiment of the method of the invention, said RNA or polypeptide is obtained from heart tissue. A suitable way would be to take a biopsy (catheter) from the ventricular wall. The decision to do this is clearly affected by the severity of the disease and the general constitution of the patient. The cardiologist and the patient have to drive the final decision. In an additionally preferred embodiment of the method of the invention, said RNA or polypeptide is quantitated in heart tissue.

In another preferred embodiment, the method of the invention further comprises the step of normalizing the amount of RNA or polypeptide against a corresponding RNA or polypeptide from a healthy subject or cells derived from a healthy subject.

The term "healthy subject" in connection with the present invention means a subject without any indication for heart disease.

The term "normalizing the amount of RNA or polypeptide against a corresponding RNA or polypeptide from a healthy subject or cells derived from a healthy subject" means, in accordance with the present invention, that levels of mRNA or polypeptide from a comparative number of cells from the heart of said subject under investigation and from the heart of an individual not affected by a disease of the heart are compared. Alternatively, cells from the heart of the subject under investigation may be compared in terms of the indicated mRNA or polypeptide levels with cells derived from the heart of a healthy individual which are kept in cell culture and optionally form a cell line. Optionally, different sources of cells such as from different individuals and/or different cell lines may be used for the generation of the standard against which the mRNA or polypeptide level of the subject under investigation is compared. Using the Affymetrix Chip technology, there is also the possibility to use external standards (that are given separately to the hybridisation cocktail) in order to normalize the values of different oligonucleotide-chips.

In yet another preferred embodiment, the method of the invention further comprises the step of normalizing the amount of polypeptide against a corresponding polypeptide from a healthy subject or cells derived from a healthy subject.

The same considerations as developed for the previous embodiment on the mRNA level apply here to the normalization of protein levels.

Additionally, the invention relates to a method for identifying a compound that increases the level in heart tissue of a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, said method comprising the steps of: (1) contacting a DNA encoding said polypeptide under conditions that would permit the translation of said polypeptide with a test compound; and (2) detecting an increased level of the polypeptide relative to the level of translation obtained in the absence of the test compound.

The term "compound" in accordance with the present invention shall mean any biologically active substance that has an effect on heart tissue or a single heart cell, whereas such compound has a positive or negative influence upon such heart tissue or heart cell. Preferred compounds are nucleic acids, preferably coding for a peptide, polypeptide, antisense RNA or a ribozyme or nucleic acids that act independently of their transcription respective their translation as for example an antisense RNA or ribozyme; natural or synthetic peptids, preferably with a relative molecular mass of about 1.000, especially of about 500, peptide analogs polypeptides or compositions of polypeptides, proteins, protein complexes, fusion proteins, preferably antibodies, especially murine, human or humanized antibodies, single chain antibodies, F_ab fragments or any other antigen binding portion or derivative of an antibody, including modifications of such molecules as for example glycosylation, acetylation, phosphorylation, famesylation, hydroxylation, methylation or estrification, hormones, organic or anorganic molecules or compositions, preferably small molecules with a relative molecular mass of about 1.000, especially of about 500.

The term "under conditions that would permit the translation of said polypeptide" denotes any conditions that allow the in vitro or in vivo translation of the polypeptide of interest. As regards in vitro conditions, translation may be effected in a cell-free system, as described, for example in Stoss, Schwaiger, Cooper and Stamm (1999). J. Biol. Chem. 274: 10951-10962, using the TNT-coupled reticulocyte lysate system (Promega). With respect to in vivo conditions, physiological conditions such as conditions naturally occurring inside a cell are preferred.

Based on the finding that expression of genes encoding the above recited polypeptides is aberrant, the method of the invention allows the convenient identification or isolation of compounds that counteract such aberrant expression such that normal expression levels are restored or essentially restored.

The DNA encoding the polypeptide of interest would normally be contained in an expression vector. The expression vectors may particularly be plasmids, cosmids, viruses or bacteriophages used conventionally in genetic engineering plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise the aforementioned polynucleotide. Preferably, said vector is a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides into targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989). Alternatively, the polynucleotides and vectors can be reconstituted into liposomes for delivery to target cells. The vectors containing the polynucleotides can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium phosphate or DEAE-Dextran mediated transfection or electroporation may be used for eukaryotic cellular hosts; see Sambrook, supra. Such vectors 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. The polynucleotide is operatively linked to expression control sequences allowing expression in eukaryotic cells. Expression of said polynucleotide comprises transcription of the polynucleotide into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and, optionally, a poly-A signal ensuring termination of transcription and stabilization of the transcript, and/or an intron further enhancing expression of said polynucleotide. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally- associated or heterologous promoter regions. Possible regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40- poly-A site or the tk-poly-A site, downstream of the polynucleotide. Furthermore, depending on the expression system used leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the aforementioned polynucleotide 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 portion thereof, into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an C- or N-terminai identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDVI

(Pharmacia), pCDM8, pRc/CMV, pcDNAI , pcDNA3, the Echo™ Cloning System (Invitrogen), pSPORTI (GIBCO BRL) or pRevTet-On/pRevTet-Off or pCI (Promega).

Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. As mentioned above, the vector used in the method of the present invention may also be a gene transfer or targeting vector. 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. Suitable vectors and methods 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; Wang, Nature Medicine 2 (1996), 714-716; W094/29469; WO 97/00957 or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and references cited therein. The polynucleotides and vectors may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell. Preferably, said cell is a germ line cell, embryonic cell, or egg cell or derived therefrom, most preferably said cell is a stem cell.

The vector comprising the DNA would be used to transform a suitable eukaryotic host cell. Upon expression of the DNA, which may be constitutive or induced, the test compound would be contacted with the DNA. This can be done by introducing the test compound into the cell. For example, if the test compound is a (poly)peptide, then introduction may be effected by transfection of the corresponding DNA, optionally comprised in a suitable expression vector. If the compound is a small molecule, preferably with a relative molecular weight of up to 1 ,000, especially up to 500, the introduction into the cell may be effected by direct administration, optionally in conjunction with DMSO for hydrophobic compounds, especially liposomal transfer.

In the case that the method of the invention is carried out in vitro, for example, in a cell-free system, then introduction into a cell would not be necessary. Rather, the test compound would be admixed to the in vitro expression system and the effect of said admixture observed.

The effect of the contact of the DNA of interest with the test compound on the protein level may be assessed by any technology that measures changes in the quantitative protein level. Such technologies include Western blots, ELISAs, RIAs and other techniques referred to herein above. The change in protein level, if any, as a result of the contact of said DNA and said test compound is compared against a standard. This standard is measured applying the same test system but omits the step of contacting the compound with the DNA. The standard may consist of the expression level of the polypeptide after no compound has been added. Alternatively, the DNA may be contacted with a compound that has previously been demonstrated to have an influence on the expression level.

Compounds tested positive for being capable of enhancing the amount of polypeptide produced are prime candidates for the direct use as a medicament or as lead compounds for the development of a medicament. Naturally, the toxicity of the compound identified and other well-known factors crucial for the applicability of the compound as a medicament will have to be tested. Methods for developing a suitable active ingredient of a pharmaceutical composition on the basis of the compound identified as a lead compound are described elsewhere in this specification.

Additionally, the invention relates to a method for identifying a compound that specifically binds to a polypeptide having an amino acid sequence selected from the group consisting of (a) the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution; said method comprising the steps of (1) providing said polypeptide; and (2) identifying a compound that is capable of binding said polypeptide.

In a preferred embodiment of the method of the invention said binding results in activation of said polypeptide. Said activation may be, for example, an enzymatic activity (as described herein below) and/or the initiation of a signal cascade.

Based on the function of these proteins in DCM development a cell based assay can be developed to identify potential activators. The protein under investigation is expressed in cardiomyocytes (e. g. by infection with recombinant adenovirus). The expression of these proteins leads to characteristic morphological alterations. Reversal or reduction of these morphological alterations can be used e.g. in an HTS assay to identifiy compounds which act as activators of these proteins. The system can be automated by use of digital image analysis systems.

Another possibility is to identify first proteins which are binding partners of the described proteins. This is especially important for structural proteins or adaptor proteins in signal transduction pathways.

Methods to identifiy compounds capable of binding include affinity chromatography with immobilised target protein and subsequent elution of bound proteins (e. g. by acid pH), co-immunoprecipitation and chemical crosslinking with subsequent analysis on SDS-PAGE.

The influence of compounds on these protein-protein interactions can be monitored by techniques like optical spectroscopy (e. g. fluorescence or surface plasmon resonance), calorimetry (isothermal titration microcalorimetry) and NMR. In the case of optical spectroscopy either the intrinsic protein fluorescence may change (in intensity and/or wavelength of emission maximum) upon complex formation with the binding compound or the fluorescence of a covalently attached fluorophore may change upon complex formation. The claimed protein or its identified binding partner may be labelled on e. g. cysteine or lysine residues with a fluorophore (for a collection of fluorophores see catalogues of Molecular Probes or Pierce Chemical Company) which changes its optical properties upon binding. These changes in the intrinsic or extrinsic fluorescence may be applied for use in a HTS assay to identifiy compounds capable of inhibiting or activating the mentioned protein-protein interaction.

If the protein referred to above exhibits enzymatic activity (e. g. Kinase, Protease, Phosphatase) the activation of this activity may be monitored by using labelled (fluorescently, radioactively or immunologically) derivates of the substrate. This activity assay which is based on labelled substrates can be used for development of a HTS assay to identifiy compounds acting as activators.

Further, the invention relates to a monoclonal antibody or derivative thereof that specifically binds to polypeptide having an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10. The invention further comprises kits comprising the monoclonal antibody or derivative thereof.

Moreover, the invention relates to a method for identifying a compound that increases the level in heart tissue of an mRNA encoding a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the aminό acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, said method comprising the steps of (1) contacting a DNA giving rise to said mRNA under conditions that would permit transcription of said mRNA with a test compound; and (2) detecting an increased level of the mRNA relative to the level of transcription obtained in the absence of the test compound.

This embodiment of the invention is very similar to the previously discussed one with the exception that here mRNA levels are detected whereas in the previous embodiment protein levels are detected. Methods of assessing RNA levels which also apply to this embodiment have been described herein above.

Furthermore, the invention relates to a transgenic non-human mammal whose somatic and germ cells comprise at least one gene encoding a functional polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, said functional polypeptide has been modified, said modification being sufficient to decrease the amount of said functional polypeptide expressed in the heart tissue of said transgenic non-human mammal, wherein said transgenic non- human mammal exhibits a disease of the heart.

A method for the production of a transgenic non-human animal, for example transgenic mouse, comprises introduction of the aforementioned polynucleotide or targeting vector into a germ cell, an embryonic cell, stem cell or an egg or a cell derived therefrom. The non-human animal can be used in accordance with a screening method of the invention described herein. Production of transgenic embryos and screening of those can be performed, e.g., as described by A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press. The DNA of the embryonal membranes of embryos can be analyzed using, e.g., Southern blots with an appropriate probe; see supra. A general method for making transgenic non-human animals is described in the art, see for example WO 94/24274. For making transgenic non-human organisms (which include homologously targeted non-human animals), embryonal stem cells (ES cells) are preferred. Murine ES cells, such as AB-1 line grown on mitotically inactive SNL76/7 cell feeder layers (McMahon and Bradley, Cell 62:1073-1085 (1990)) essentially as described (Robertson, E. J. (1987) in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E. J. Robertson, ed. (Oxford: IRL Press), p. 71-112) may be used for homologous gene targeting. Other suitable ES lines include, but are not limited to, the E14 line (Hooper et al., Nature 326:292-295 (1987)), the D3 line (Doetschman et al., J. Embryol. Exp. Morph. 87:27-45 (1985)), the CCE line (Robertson et al., Nature 323:445-448 (1986)), the AK-7 line (Zhuang et al., Cell 77:875-884 (1994)). The success of generating a mouse line from ES cells bearing a specific targeted mutation depends on the pluripotence of the ES cells (i. e., their ability, once injected into a host developing embryo, such as a blastocyst or morula, to participate in embryogenesis and contribute to the germ cells of the resulting animal). The blastocysts containing the injected ES cells are allowed to develop in the uteri of pseudopregnant nonhuman females and are born as chimeric mice. The resultant transgenic mice are chimeric for cells having either the recombinase or reporter loci and are backcrossed and screened for the presence of the correctly targeted transgene (s) by PCR or Southern blot analysis on tail biopsy DNA of offspring so as to identify transgenic mice heterozygous for either the recombinase or reporter locus/loci.

The transgenic non-human animals may, for example, be transgenic mice, rats, hamsters, dogs, monkeys, rabbits, pigs, or cows. Preferably, said transgenic non- human animal is a mouse.

In a preferred embodiment of the transgenic non-human mammal of the invention said functional or disrupted gene was introduced into the non-human mammal or an ancestor thereof, at an embryonic stage.

In a further preferred embodiment of the transgenic non-human mammal of the invention the modification is inactivation or suppression of said gene(s) or leads to the reduction of the synthesis of the corresponding protein(s).

This embodiment allows for example the study of the interaction of various mutant forms of the aforementioned polypeptides on the onset of the clinical symptoms of a disease related to disorders in the heart. All the applications that have been herein before discussed with regard to a transgenic animal also apply to animals carrying two, three or more transgenes for example encoding different aforementioned nucleic acid molecules. It might be also desirable to inactivate protein expression or function at a certain stage of development and/or life of the transgenic animal. This can be achieved by using, for example, tissue specific, developmental and/or cell regulated and/or inducible promoters which drive the expression of, e.g., an antisense or ribozyme directed against the RNA transcript encoding the corresponding RNA; see also supra. A suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. 89 USA (1992), 5547-5551) and Gossen et al. (Trends Biotech. 12 (1994), 58-62). Similar, the expression of the mutant protein(s) may be controlled by such regulatory elements.

As mentioned, the invention also relates to a transgenic non-human animal, preferably mammal and cells of such animals which cells contain (preferably stably integrated into their genome) at least one of the aforementioned nucleic acid molecule(s) or part thereof, wherein the transcription and/or expression of the nucleic acid molecule or part thereof leads to reduction of the synthesis of (a) corresponding protein(s). In a preferred embodiment, the reduction is achieved by an anti-sense, sense, ribozyme, co-suppression and/or dominant mutant effect. "Antisense" and "antisense nucleotides" means DNA or RNA constructs which block the expression of the naturally occurring gene product.

Techniques how to achieve this are well known to the person skilled in the art. These include, for example, the expression of antisense-RNA, ribozymes, of molecules which combine antisense and ribozyme functions and/or of molecules which provide for a co-suppression effect; see also supra. When using the antisense approach for reduction of the amount of said proteins in cells, the nucleic acid molecule encoding the antisense-RNA is preferably of homologous origin with respect to the animal species used for transformation.

However, it is also possible to use nucleic acid molecules which display a high degree of homology to endogenously occurring nucleic acid molecules encoding such a protein. In this case the homology is preferably higher than 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99%.

In cases where more than one of the aforementioned genes are inactivated, interrelationships of gene products in the onset or progression of the diseases of the heart may be assessed. In this regard, it is also of interest to cross transgenic non-human animals having different transgenes for assessing further interrelationships of gene products in the onset or progression of said disease. Consequently, the offspring of such crosses is also comprised by the scope of the present invention.

In addition, the invention relates to a method for identifying a compound that increases the expression of a polypeptide in heart tissue selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, said method comprising the steps of: (1) contacting a transgenic non-human mammal as described herein above with a test compound, and (2) detecting an increased level of expression of said polypeptide relative to the expression in the absence of said test compound.

The test compound which has preferably been tested beforehand for essentially lacking toxicity for the animal can be administered to the animal by any convenient route suitable for administration. These routes include injection, topical and oral administration. Intervals and doses of administration may vary and will be decided upon by the physician/researcher on a case-by-case basis.

Detection, if any, may be effected by a variety of means. For example, if the transgene includes a bioluminescent portion, increase of polypeptide production may be assessed as described, for example, in EP 95 94 1424.4 or in EP 99 12 4640.6. Alternatively, and if the polypeptides are present in the bloodstream, blood of the non-human transgenic animal may be assessed for the changing quantity of the protein. It is preferred in such a case that the gene encoding the polypeptide of interest carries an inducible promoter. Thus, by comparing the situations with and without induction, it can conveniently be determined whether the test compound has indeed an effect on the polypeptide produced or whether the test compound causes an effect unrelated to the level of polypeptide produced. In certain embodiments of the invention, the non-human transgenic animal will have to be sacrificed in order to assess whether a change in the level of polypeptide expression has occurred. For example, heart tissue may be removed from the sacrificed animal and assessed, using standard technologies, for the expression level of the protein. For example, an antibody specific for the polypeptide may be contacted with the heart tissue and the test developed with a second labeled antibody that is directed to the first antibody. Alternatively, the first antibody itself may be labeled. Heart tissue of a non-human transgenic animal that has been contacted with the test compound would be compared with heart tissue of a non- human transgenic animal that has not been contacted with said test compound.

As mentioned herein above, the transgenic animal may carry more than one of the aforementioned nucleic acid molecules. Accordingly, the effect of a test compound on the expression level of any of these transgenes may be assessed. In addition, a variety of test compounds may be tested, at the same time, for the effect on one or a variety of said transgenes.

A test compound that has proven to be effective in increasing the level of the polypeptide of interest and/or in decreasing the turnover of the polypeptide of interest may be either directly formulated into a medicament (if, for example, its structure is suitable for administration and if it has proven to be non-toxic) or may serve as a lead compound for downstream developments, the results of which may then be formulated into pharmaceutical compositions.

In a preferred embodiment of the method of the invention the test compound prevents or ameliorates a disease of the heart in said transgenic non-human mammal.

In this embodiment, the effect of the test compound may be assessed by observing the disease state of the transgenic animal. Thus, if the animal suffers from a disease of the heart prior to the administration of the test compound and the administration of the test compound results in an amelioration of the disease, then it can be concluded that this test compound is a prime candidate for the development of a medicament useful also in humans. In addition the compound could also inhibit disease establishment by treatment in advance. A further embodiment of the invention is a method for identifying one or a pluratiy of isogenes of a gene coding for a polypeptide selected from the group consisting of: the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; in heart tissue comprising the steps of (1) providing nucleic acid coding for said polypeptide or a part thereof; and (2) identifying a second nucleic acid from the same species that (i) has a homology of 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% or (ii) hybridizes in 4xSSC, 0.1 SDS at 45°C to the nucleic acid molecule encoding said amino acid sequences.

The term "isogenes" shall mean genes that are considered to be generated by gene duplication. They can be identified by comparing the homology of the DNA-, RNA-, or protein-sequence of interest with other DNA, RNA or protein-sequences of the same species. There might be strong differences in the degree of homology between isogenes of the same species. This may be dependent on the time-point, when the gene duplication event took place in evolution and the degree of conservation during evolution.

Isogenes can be identified and cloned by RT-PCR as has been demonstrated by Screaton et al. (1995) EMBO J. 14:4336-4349 or Huang et al. (1998) Gene 211 : 49-55. Isogenes can also be identified and cloned by colony hybridisation or plaque hybridization as described in Sambrook, Fritsch, Maniatis (1989), Molecular Cloning. Cold Spring Harbor Laboratory Press. In a first step, either a genomic or a cDNA library e.g. in bacteria or phages is generated. In order to identify isogenes, colony hybridisation or plaque hybridization is slightly modified in a way that cross- hybridizations are detectable under conditions of lower stringency. This can be achieved by lowering the calculated temperature for hybridisation and washing and/or by lowering the salt concentration of the washing solutions (Sambrook, Fritsch, Maniatis (1989) Cold Spring Harbor Laboratory Press). For example, a low-stringency washing condition may include 2 washing steps at a temperature between 45°C and 65°C with 4xSSC, 0,1% SDS for 30 min (50 ml) and finally two washing steps with 50 ml of a solution containing 2xSSC, 0.1% SDS for 30 min. After detection, signal intensity of colonies containing an isogene is dependent on the homology of a gene and its isogene(s).

Furthermore, the invention relates to a method for identifying one or a plurality of genes whose expression in heart tissue is modulated by inhibiting or reducing the expression of a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, or of an mRNA encoding said polypeptide, said modulation being indicative of a disease of the heart, said method comprising the steps of: (1) contacting a plurality of heart tissue cells with a compound that inhibits or decreases the expression of said polypeptide under conditions that permit the expression of said polypeptide in the absence of a test compound, and (2) comparing a gene expression profile of said heart cell in the presence and in the absence of said compound.

The term "gene expression profile" shall mean all expressed genes of a cell or a tissue. Such profile can be assessed using methods well known in the art, for example isolation of total RNA, isolation of poly(A) RNA from total RNA, suppression subtractive hybridization, differential display, preparation of cDNA libraries or quantitative dot blot analysis, as for example described in Example 1 of this specification.

This embodiment of the method of the invention is particularly suitable for identifying further genes the expression level of which is directly affected by the aberrant expression of any of the aforementioned genes. In other words, this embodiment of the method of the invention allows the identification of genes involved in the same protein cascade as the aberrantly expressed gene. Typically, the method of the invention will be a method performed in cell culture.

The method of the invention allows for the design of further medicaments that use other targets than the aberrantly expressed gene. For example, if a potential target downstream of the aberrantly expressed gene is indeed targeted by a medicament, the negative effect of the aberrantly expressed gene may be efficiently counterbalanced. Compounds modulating other genes in the cascade may have to be refined or further developed prior to administration as a medicament as described elsewhere in this specification.

Additionally, the invention relates to a method for identifying one or a plurality of genes whose expression in heart tissue is modulated by the inhibition or reduction of the expression of a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the arnino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, or of an mRNA encoding said polypeptide, said modulation being indicative of a disease of the heart, said method comprising the steps of: (1) providing expression profiles of (i) a plurality of heart tissue cells from or derived from a heart of a subject suffering from a disease of the heart; and (ii) a plurality of heart tissue cells from or derived from a subject not suffering from a disease of the heart; and (2) comparing the expression profiles (i) and (ii).

In variation to the method described herein above, this embodiment of the method of the invention compares the expression profiles of cells from a healthy subject and a subject suffering from a heart disease. In this regard, the term "cells derived from a heart" includes cells that are held in cell culture or even cell lines that autonomously grow in cell culture and that were originally derived from heart tissue. By comparing the two expression profiles, differences in expression levels of genes involved in the disease of the heart may be identified. As with the preceding embodiment, these genes may be part of a cascade involving the aberrantly expressed gene. Examples of such cascades are signaling cascades. Once genes are identified that are expressed at a different level in a diseased heart, they may be tested up-regulation or down-regulation by bringing them into contact with suitable test compounds. Again, these test compounds may then, with or without further development, be formulated into pharmaceutical compositions.

In a preferred embodiment, the method of the invention further comprises the steps of (3) determining at least one gene that is expressed at a lower or higher level in the presence of said compound; and (4) identifying a further compound that is capable of raising or lowering the expression level of said at least one gene.

This preferred embodiment of the invention requires that one of the genes the expression of which may directly or indirectly be lowered or increased by the expression of the aberrant gene is identified. Then, a further panel of test compounds may be tested for the capacity to increase or decrease the expression of said further gene. Compounds that are successfully tested would be prime candidates for the development of medicaments for the prevention or treatment of a disease of the heart.

In another preferred embodiment, the method of the invention further comprises the steps of (3) determining at least one gene that is expressed at a lower or higher level in said heart tissue cells from or derived from a heart of a subject suffering from a disease of the heart; and (4) identifying a further compound that is capable of raising or lowering the expression level of said at least one gene.

In variation of the previously discussed embodiment, this embodiment requires that at least one gene is identified by comparing the expression profiles of tissue or cells derived from a healthy subject and from a subject suffering from a disease of the heart. Subsequently, at least one compound is identified that is capable of increasing or decreasing the expression of said gene.

Additionally, the invention relates to a method for identifying proteins or a plurality of proteins in heart tissue whose activity is modulated by a polypeptide having the amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; said method comprising the steps of (1) providing said polypeptide and (2) identifying a further protein that is capable of interacting with said polypeptide.

One possible method to identify protein-protein interactions is the Yeast two-hybrid screen described by Golemis & Khazak (1997), Methods Mol Biol. 63:197-218. Other well established methods in order to identify protein-protein interactions are co-immunoprecipitations or in vitro protein interaction assays like GST-pulldown assays (such as described in Stoss, Schwaiger, Cooper and Stamm (1999). J. Biol. Chem. 274: 10951-10962). In a further preferred embodiment of the method of the invention said compound is a small molecule or a peptide derived from an at least partially randomized peptide library.

Additionally, the invention relates to a method of refining a compound identified by the method as described herein above, said method comprising the steps of (1) identification of the binding sites of the compound and the DNA or mRNA molecule by site-directed mutagenesis or chimeric protein studies; (2) molecular modeling of both the binding site of the compound and the binding site of the DNA or mRNA molecule; and (3) modification of the compound to improve its binding specificity for the DNA or mRNA.

This method, in a preferred embodiment comprises the identification steps of the method described herein above, prior to the refinement steps.

All techniques employed in the various steps of the method of the invention are conventional or can be derived by the person skilled in the art from conventional techniques without further ado. Thus, biological assays based on the herein identified nature of the polypeptides may be employed to assess the specificity or potency of the drugs wherein the increase of one or more activities of the polypeptides may be used to monitor said specificity or potency. Steps (1) and (2) can be carried out according tor conventional protocols. A protocol for site directed mutagenesis is described in Ling MM, Robinson BH. (1997) Anal. Biochem. 254: 157-178. The use of homology modelling in conjunction with site-directed mutagenesis for analysis of structure-function relationships is reviewed in Szklarz and Halpert (1997) Life Sci. 61 :2507-2520. Chimeric proteins are generated by ligation of the corresponding DNA fragments via a unique restriction site using the conventional cloning techniques described in Sambrook, Fritsch, Maniatis. Molecular Cloning, a laboratory manual. (1989) Cold Spring Harbor Laboratory Press. A fusion of two DNA fragments that results in a chimeric DNA fragment encoding a chimeric protein can also be generated using the gateway-system (Life technologies), a system that is based on DNA fusion by recombination. A prominent example of molecular modelling is the structure-based design of compounds binding to HIV reverse transcriptase that is reviewed in Mao, Sudbeck, Venkatachalam and Uckun (2000). Biochem. Pharmacol. 60: 1251-1265.

For example, identification of the binding site of said drug by site-directed mutagenesis and chimerical protein studies can be achieved by modifications in the (poly)peptide primary sequence that affect the drug affinity; this usually allows to precisely map the binding pocket for the drug.

As regards step (2), the following protocols may be envisaged: Once the effector site for drugs has been mapped, the precise residues interacting with different parts of the drug can be identified by combination of the information obtained from mutagenesis studies (step (1)) and computer simulations of the structure of the binding site provided that the precise three-dimensional structure of the drug is known (if not, it can be predicted by computational simulation). If said drug is itself a peptide, it can be also mutated to determine which residues interact with other residues in the polypeptide of interest.

Finally, in step (3) the drug can be modified to improve its binding affinity or ist potency and specificity. If, for instance, there are electrostatic interactions between a particular residue of the polypeptide of interest and some region of the drug molecule, the overall charge in that region can be modified to increase that particular interaction.

Identification of binding sites may be assisted by computer programs. Thus, appropriate computer programs can be used for the identification of interactive sites of a putative inhibitor and the polypeptide by computer assisted searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 114-120). Further appropriate computer systems for the computer aided design of protein 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. Modifications of the drug can be produced, for example, by peptidomimetics and other inhibitors can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds. 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 activators of the expression of the polypeptide of the invention can be used for the design of peptidomimetic activators, e.g., in combination with the (poly)peptide of the invention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).

In accordance with the above, in a preferred embodiment of the method of the invention said compound is further refined by peptidomimetics.

The invention furthermore relates to a method of modifying a compound identified or refined by the method of the invention as described herein above as a lead compound to achieve (i) modified site of action, spectrum of activity, organ specificity, and/or (ii) improved potency, and/or (iii) decreased toxicity (improved therapeutic index), and/or (iv) decreased side effects, and/or (v) modified onset of therapeutic action, duration of effect, and/or (vi) modified pharmakinetic parameters (resorption, distribution, metabolism and excretion), and/or (vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or (viii) improved general specificity, organ/tissue specificity, and/or (ix) optimized application form and route by (i) esterification of carboxyl groups, or (ii) esterification of hydroxyl groups with carbon acids, or (iii) esterification of hydroxyl groups to, e.g. phosphates, pyrophosphates or sulfates or hemi succinates, or (iv) formation of pharmaceutically acceptable salts, or (v) formation of pharmaceutically acceptable complexes, or (vi) synthesis of pharmacologically active polymers, or (vii) introduction of hydrophylic moieties, or (viii) introduction/exchange of substituents on aromates or side chains, change of substituent pattern, or (ix) modification by introduction of isosteric or bioisosteric moieties, or (x) synthesis of homologous compounds, or (xi) introduction of branched side chains, or (xii) conversion of alkyl substituents to cyclic analogues, or (xiii) derivatisation of hydroxyl group to ketales, acetales, or (xiv) N-acetylation to amides, phenylcarbamates, or (xv) synthesis of Mannich bases, imines, or (xvi) transformation of ketones or aldehydes to Schiffs bases, oximes, acetales, ketales, enolesters, oxazolidines, thiozolidines or combinations thereof. The method of modifying a compound identified or refined optionally comprises in accordance with the present invention (the) steps of the methods described herein above.

The various steps recited above are generally known in the art. They include or rely on quantitative structure-action relationship (QSAR) analyses (Kubinyi, "Hausch-Analysis and Related Approaches", VCH Verlag, Weinheim, 1992), combinatorial biochemistry, classical chemistry and others (see, for example, Holzgrabe and Bechtold, Deutsche Apotheker Zeitung 140(8), 813-823, 2000).

The invention additionally relates to a method for inducing a disease of the heart in a non-human mammal, comprising the step of contacting the heart tissue of said mammal with a compound that inhibits or reduces/decreases the expression and/or activity of a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ1D NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution.

This embodiment of the invention is particularly useful for mimicking factors/developments leading to the onset of the disease. The fact, that differences in the expression of a protein contributes to heart failure has been shown for phospholamban, for example. Mice over-expressing phospholamban develop heart failure. This effect is thought to be due to the inhibition of Serca. (Minamisawa et al. (1999) Cell, 99:313-322).

In a preferred embodiment of the method of the invention said compound that decreases or increases is a small molecule, an antibody or an aptamer that specifically binds said polypeptide.

The terms "small molecule" as well as "antibody" have been described herein above and bear the same meaning in connection with this embodiment.

The invention moreover relates to a method of producing a pharmaceutical composition comprising formulating the compound identified, refined or modified by the method as described herein above optionally with a pharmaceutically active carrier and/or diluent. Said method of producing a pharmaceutical composition preferably comprises in accordance with the present invention (the) steps of the methods described herein above.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and/or diluent. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 μg (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 106 to 1012 copies of the DNA molecule. The compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents such as interteukins or interferons depending on the intended use of the pharmaceutical composition.

The invention also relates to a method for preventing or treating a disease of the heart in a subject in need of such treatment, comprising the step of increasing the level of a polypeptide in the heart tissue of a subject, said polypeptide being selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, in the heart tissue of the subject.

Further, the invention relates to a method of preventing or treating a disease of the heart in a subject in need of such treatment comprising the step of increasing the level of mRNA encoding a polypeptide in the heart tissue of a subject, said polypeptide being selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution.

The invention in a preferred embodiment relates to a method wherein such increase is effected by administering the pharmaceutical composition obtained by the method as described herein above.

In a further preferred embodiment of the method of the invention such an increase is effected by introducing the nucleic acid sequence, preferably DNA sequence, recited herein above into the germ line or into somatic cells of a subject in need thereof. Technologies for effecting such an introduction have been described herein above.

In a most preferred embodiment of the method of the invention the disease of the heart to be treated is congestive heart failure, dilative cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, specific heart muscle disease, rhythm and conduction disorders, syncope and sudden death, coronary heart disease, systemic arterial hypertension, pulmonary hypertension and pulmonary heart disease, valvular heart disease, congenital heart disease, pericardial disease or endocarditis.

In addition, the invention relates to a method for identifying subjects at risk for congestive heart failure comprising the step of detecting an decreased diphosphoinositol polyphosphate phosphodihydrolase type II beta (DIPP2β) activity in the heart tissue of a subject.

Moreover, the invention relates to a methocf identifying a subject at risk for congestive heart failure, said method comprising detecting increased levels of β- phosphorylated diphosphoinositol polyphosphates. A method of detecting increased levels of β-phosphorylated diphosphoinositol polyphosphates would be the structural analysis of diphosphoinositol polyphosphates in biological systems, which relies largely on NMR analysis as described in Laussmann, Eujen, Weisshuhn, Thiel and Vogel (1996) Biochem. J. 315:715-720. Another method of analysis would be the detection and quantitation of different pools of phosphorylated diphosphoinositol polyphosphates by microbore metal-dye- detection HPLC as described in Albert, Safrany, Bembenek, Reddy, Reddy, Falck, Brockner, Shears and Mayr (1997) Biochem. J. 327: 553-560. The invention additionally relates to a method for preventing or treating congestive heart failure in a subject, said method comprising the step of contacting the heart tissue of said subject with a compound that increases the expression and/or the activity of DIPP2β or DIPP2 .

The invention as well relates to a method for preventing or treating congestive heart failure in a subject, said method comprising the step of increasing the removal of β-phosphate from diphosphoinositol polyphosphates in the heart tissue of said subject.

The invention in a preferred embodiment relates to a method wherein the expression of DIPP2β is increased in said heart tissue.

In- a particularly preferred embodiment of the method of the invention the expression of DlPP2 is increased in said heart tissue.

In a further particularly preferred embodiment of the method of the invention the expression of an exogenous gene is increased in said heart tissue.

In a further particularly preferred embodiment of the method of the invention the expression of an endogenous gene is increased in said heart tissue.

In a preferred embodiment of the method of the invention the activity of DIPP2β is increased in said heart tissue.

In a further preferred embodiment of the method of the invention the activity of DIPP2α is increased in said heart tissue.

The invention further relates to a method for preventing or treating congestive heart failure in a subject comprising the step of decreasing the amount of β- phosphorylated diphosphoinositol polyphosphates in the heart tissue of said subject.

The invention additionally relates to a method for identifying a compound for preventing or treating congestive heart failure, said method comprising the steps of: (a) contacting DIPP2β or DIPP2 with a substrate for DIPP2β or DIPP2α and a test compound, and (b) determining whether the removal of β-phosphate from the substrate is increased in the presence of the test compound.

The invention as well relates to a method for identifying a compound for preventing or treating congestive heart failure, said method comprising the steps of: (a) contacting a heart tissue cell expressing DIPP2β or DIPP2α with a test compound; and (b) determining whether removal of β-phbsphate from diphosphoinositol polyphosphates is increased.

Additionally, the invention relates to a method for preventing or treating congestive heart failure in a subject comprising the step of increasing the glucose level in heart tissue in said subject.

In a preferred embodiment of the method of the invention said glucose level is increased by administering glucose to said subject.

In another preferred embodiment of the method of the invention said glucose level is increased by increasing the amount of glycogen storage in said heart tissue.

In a particularly preferred embodiment of the method of the invention glycogen storage is increased by increasing the expression and/or activity of a glucan branching enzyme (GBE). The activity of alpha-1,4-glucan branching enzyme can be measured as described in Satoh and Sato (1980). Anal. Biochem. 108: 16-24 or in Tolmasky and Krisman (1987) Eur. J. Biochem. 168: 393-397 and references therein. In a most preferred embodiment of the method of the invention the GBE is 1,4-α- GBE.

In addition, the invention relates to a method for preventing or treating congestive heart failure in a subject, comprising increasing the activity of DERP2.

Moreover, the invention relates to a method for preventing or treating congestive heart failure in a subject, comprising increasing the DERP2-mediated signal transduction in the heart tissue of said subject.

The invention in addition relates to a method for preventing or treating congestive heart failure in a subject, comprising increasing DERP2-mediated cellular transport in the heart tissue of said subject.

In a preferred embodiment of the invention, the method comprises the step of increasing the expression of DERP2 on the plasma membrane of cells in said heart tissue.

In an additionally preferred embodiment of the invention, the method comprises the step of contacting said heart tissue with a compound that is a DERP2 activator.

In a further embodiment the invention relates to the use of a compound identified, refined or modified by the method as described herein above or a monoclonal antibody for the manufacture of a pharmaceutical composition for the prophylaxis or treatment of heart diseases, especially congestive heart failure.

The figures show:

Fig. 1 A shows the cDNA sequence of clone 40095.

Fig. 1 B shows the sequence of the EST clone AF047439. Start and stop codons are marked by bold letters, the sequence of 40095 is marked in italic letters. The putative polyadenylation signal is underlined.

Fig. 1 C shows the putative amino acid sequence AAC39890. Fig.1 D shows a schematic alignment of the cDNA fragment 40095 identified in SSH with the homologous Genbank entree and the open reading frame of 300 amino acids (aa). Not to scale.

Fig. 1 E: Two filters were hybridized sequentially with [α-³³P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and four DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given. Mean values and standard deviations were calculated from all NF and DCM samples, respectively. Asterisks mark samples used for SSH.

Fig. 2 A shows the cDNA sequence of clone 41237.

Fig. 2 B shows the sequence of 41237contig (EST sequences available from proprietary LEADS™ database by Compugen Ltd. have been assembled using

LabOnWeb algorithms). Start and stop codons are marked in bold letters, the sequence of 41237 is given in italic letters.

Fig. 2 C shows the amino acid sequence AAF68859. The amino acid sequence of the beta isoform is given. The deletion of Gln86 (underlined) within the type 2 alpha coding sequence results in a 20-50% increase in catalytic activity of the alpha isoform.

Fig.2 D: Schematic alignment of the cDNA fragment 41237 identified in SSH with its overlapping contig of assembled ESTs according to LabOnWeb analysis

(Compugen), homologous Genbank entrees and the open reading frame of 181 amino acids (aa). Not to scale.

Fig. 2 E: Two filters were hybridized sequentially with [ -³³P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and four DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%), relative expression levels are given.

Mean values and standard deviations were calculated from all NF and DCM samples, respectively. Asterisks mark samples used for SSH.

Fig.3 A shows the cDNA sequence of clone 41444. Fig. 3 B shows the sequence of the EST clone L07956. Start and stop codons are marked in bold letters, the sequence of 41444 is given in italic letters. Putative polyadenylation sites are underlined.

Fig. 3 C shows the amino acid sequence AAA58642.

Fig.3 D: Schematic alignment of the cDNA fragment 41444 identified in SSH with its homologous Genbank entree and the open reading frame of 702 amino acids

(aa). Not to scale.

Fig. 3 E: Two filters were hybridized sequentially with [ -³³P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and four DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given.

Fig. 3 F: The figure shows the hybridization pattern of GBE on HG-U133A oligo arrays (Affymetrix) in different tissues samples and heart RNA mixture. Individual relative signal intensities as calculated using MAS5 software of Affymetirx and relative ratios between tissues are given.

Fig. 4 A shows the cDNA sequence of clone 41482.

Fig. 4 B shows the sequence of the EST clone AB009685. Start and stop codons are marked in bold letters, the sequence of 41482 is marked in italic letters.

Fig. 4 C shows the putative amino acid sequence BAA93049.

Fig.4 D: Schematic alignment of the cDNA fragment 41482 identified in SSH with its homologous Genbank entrees and putative open reading frames of 345 and 319 amino acids (aa), respectively. Not to scale.

Fig. 4 E: Two filters were hybridized sequentially with [α-³³P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and four DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given.

Mean values and standard deviations were calculated from all NF and DCM samples, respectively. Asterisks mark samples used for SSH. Fig. 4 F: The figure shows the sequence of 41482contig, which has been assembled from AB009685 and the cDNA fragment identified by SSH.

Fig. 5 A shows the cDNA sequence of clone 52529.

Fig. 5 B shows the sequence of the EST clone NM_006854. Start and stop codons are marked by bold letters, the sequence of 52529 is marked in italic letters.

Fig. 5 C shows the putative amino acid sequence NPJ 06845.

Fig.5 D: Schematic alignment of the cDNA fragment 52529 identified in SSH with its homologous Genbank entree and the open reading frame of 212 amino acids

(aa). Not to scale.

Fig. 5 E: Two filters were hybridized sequentially with [ -³³P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and five DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given.

Fig. 6 A shows the cDNA sequence of clone 55253.

Fig. 6 B shows the the sequence of the EST clone U06631. Start and stop codons are marked by bold letters, the sequence of 55253 is marked in italic letters. Two putative polyadenylation sites are underlined.

Fig. 6 C shows the amino acid sequence AAA16607.

Fig.6 D: Schematic alignment of the cDNA fragment 55253 identified in SSH with its homologous Genbank entree and the open reading frame of 597 amino acids

(aa). Not to scale.

Fig. 6 E: Two filters were hybridized sequentially with [ -³³P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and five DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given.

Mean values and standard deviations were calculated from all NF and DCM samples, respectively.

Fig. 7 A shows the cDNA sequence of clone 61024. Fig. 7 B shows the sequence of the EST clone 61024cons. The stop codon is marked by bold letters, the start codon is not annotated so far. The sequence of

61024 is marked in italic letters.

Fig. 7 C shows the sequence of the 5^' end of 61024 elongated by 292 nt as a result of PCR. The start methionine was still not identified and seems to be further upstream.

Fig. 7 D shows the putative amino acid sequence 61024pep. The protein sequence of BAA82992 could be elongated at the 5^Λ end by 98 amino acids as underlined.

Fig.7 E: Schematic alignment of the cDNA fragment 61024 identified in SSH with the homologous Genbank entree. The open reading frame identified so far comprises 642 amino acids (aa). Not to scale.

Fig. 7 F: Two filters were hybridized sequentially with [α-³³P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and five DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given.

Fig. 8 A shows the cDNA sequence of clone 61119.

Fig. 8 B shows the sequence of the EST clone AF115509. Start and stop codons are marked by bold letters, the sequence of 61119 is marked in italic letters.

Fig. 8 C shows the putative amino acid sequence AAD41257.

Fig.8 D: Schematic alignment of the cDNA fragment 61119 identified in SSH with its homologous Genbank entree and the open reading frame of 721 amino acids

(aa). Not to scale.

Fig. 8 E: Two filters were hybridized sequentially with [ -³³P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and five DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given.

Mean values and standard deviations were calculated from all NF and DCM samples, respectively. Asterisks mark samples used for SSH. Fig. 9 A shows the cDNA sequence of clone 62139.

Fig. 9 B shows the sequence of 62139contig (EST sequences available from proprietary LEADS™ database by Compugen Ltd. have been assembled using

LabOnWeb algorithms). Start and stop codons are marked by bold letters, the sequence of 62139 is marked in italic letters. The putative polyadenylation signal is underlined.

Fig. 9 C shows the putative amino acid sequence CAA65989.

Fig.9 D: Schematic alignment of the cDNA fragment 62139 identified in SSH with its overlapping contig of assembled EST sequences according to LabOnWeb analysis (Compugen), homologous Genbank entree and the open reading frame of

437 amino acids (aa). Not to scale.

Fig. 9 E: Two filters were hybridized sequentially with [α-³³P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and five DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given.

Fig. 9 F: Affymetrix arrays were hybridized with biotinylated RNA isolated from either 11 DCM-, 9 ICM- or 10 non-failing tissue-samples. Normalization and data analysis occurred using the Microarray Suite 4.0 (Affymetrix). The y-axis shows the average differences measured.

Fig. 9 G: Affymetrix arrays were hybridized with biotinylated RNA isolated from either 11 DCM-, 9 ICM- or 10 non-failing tissue-samples (see FIG. 9 F). Mean values and standard deviations were calculated from all NF, DCM and ICM samples, respectively. The y-axis shows the average differences measured.

Fig. 10 A shows the cDNA sequence of clone 62140 and 58185.

Fig. 10 B shows the sequence of 62140contig (EST sequences available from proprietary LEADS™ database by Compugen Ltd. have been assembled using

LabOnWeb algorithms). The sequences of 62140 is marked in italic letters.

Fig.10 C: Schematic alignment of cDNA fragments 62140 and 58185 identified in

SSH with its overlapping contig of assembled EST sequences according to LabOnWeb (Compugen) analysis and accession numbers of homologous Genbank entrees. Not to scale.

Fig. 10 D: Two filters were hybridized sequentially with [α-³³P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and five DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given. Mean values and standard deviations were calculated from all NF and DCM samples, respectively. Asterisks mark samples used for SSH.

Examples

The following examples illustrate the invention. These examples should not be construed as limiting: the examples are included for purposes of illustration and the present invention is limited only by the claims.

EXAMPLE 1

1. Isolation of total RNA from heart tissue

Total RNA was isolated from tissue of explanted hearts of left ventricle of human non-failing and DCM patients, which are listed in TABLE 1 , respectively, according to the protocol of Chomczynski and Sacchi (1987) with some minor modifications. 0.5 g tissue were disrupted using a mortar and pestle and grinded under liquid nitrogen. The suspension of tissue powder and liquid nitrogen was decanted into a cooled 50 ml polypropylene tube and nitrogen allowed to evaporate completely without thawing the sample. After addition of 10 ml solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate pH 7.0, 0.5 % sodium-N-lauroyi-sarcosinat, 0.1 M 2-mercaptoethanol) the sample was homogenized immediately using a rotor- stator homogenizer (Ultra-Turrax T8, IKA Labortechnik) for 60 s at maximum speed. The sample was mixed with 1 ml 2 M NaOAc pH 4.0, 10 ml phenol (water saturated, pH 4.5-5) and 2 ml chloroform/isoamylalcohol (49/1). After incubation on ice for 15 min and centrifugation at 10000g for 30 min at 4 °C the aqueous phase was transferred to a fresh 50 ml polypropylene tube. RNA was precipitated with 1 vol isopropanol at -20 °C for at least one hour. After centrifugation at 10000g for 30 min at 4 °C the RNA pellet was redissolved in 5 ml solution D and precipitated again with 1 vol isopropanol as described. The pellet was washed with cold 75% EtOH and dried at RT for 15 min. To completely dissolve RNA 500 μl DEPC- treated water were added and the sample was incubated at 60 °C for 10 min, final storage was at -80 °C. An aliquot was used for quantification by A260 measurement and separation on a formaldehyde agarose gel (Sambrook et al.) to check integrity and size distribution. TABLE 1: Human heart samples

2. Isolation of poly(A) RNA from total RNA

Poly(A) RNA was isolated from 300 μg total RNA (1. supra) using the PolyA Quick mRNA Isolation Kit (Stratagene) according to the manufacturers protocol. Purified mRNA was dissolved in 30 μl RNase-free water (Stratagene), quantified and analyzed on a formaldehyde agarose gel as described (1. supra).

3. Suppression subtractive hybridization (SSH) 3.1 Construction of a subtracted library

2 μg of tester mRNA and 2 μg of driver mRNA were used to construct a subtracted and normalized cDNA library using the PCR-Select cDNA Subtraction Kit and Advantage cDNA-Polymerase Mix (Clontech) according to the manufacturers protocol. In general, two libraries were constructed for each tester and driver combination, since only transcripts can be identified that are over-represented in the tester mRNA.

Both, the subtracted and non-subtracted cDNA population were analyzed on an agarose gel as described (Clontech) and transferred onto Zeta-Probe GT nylon membrane (BioRad) by capillary forces (Sambrook et al., supra). The membrane was UV crosslinked in a Stratalinker 2400 (Stratagene).

To analyze the subtraction efficiency the membrane was hybridized with a Digoxigenin-labeled probe synthesized from a housekeeping gene using the Dig- DNA Labeling and Detection Kit (Roche). For probe synthesis a 451 bp fragment of human GAPDH was amplified from 0.5-1 μg cDNA of a NF heart library (5.1.) in a 100 μl PCR reaction with the primer pair provided by the PCR-Select cDNA Subtraction Kit (Clontech). 100 ng of gel purified (QIAquick Gel Extraction Kit, Qiagen) GAPDH cDNA fragment then were used for Dig-labeling. The hybridized membrane was exposed to a X-ray film (X OMAT AR, Kodak) for 15 min. Only subtractions, where the GAPDH signal intensity of the subtracted cDNA population was at least four fold lowered compared to the corresponding non-subtracted cDNA-population, were selected for further analysis. 17 μl of the subtracted sample were purified using a PCR Purification Kit (Qiagen) and eluted in 20 μl ddH₂θ (Gibco BRL).

For addition of 3'-A overhangs 15.7 μl of the purified subtracted cDNA sample was incubated in the presence of PCR buffer, 1.5 U Taq DNA polymerase (APB) and 0.2 mM dATP for 11 min at 72 °C. 3 μl of the sample was ligated into the pGEM-T easy vector (Promega) and competent E. coli cells were transformed as described by the manufacturer.

3.2 Amplification of subtracted cDNA clones

Subtracted cDNA clones were grown over night at 37 °C in 96 well microplates filled with 100 μl LB medium (Sambrook et al. , supra.) and supplemented with 10 μg/ml Amp. 1 μl of the bacterial culture then was transferred into 99 μl PCR premix (1x PCR buffer, 2.5 U Taq DNA polymerase (APB), 0.2 mM dNTP) and directly amplified using the nested primer pair 1 and 2R provided by the PCR-Select cDNA Subtraction Kit (Clontech). Best results were obtained with 27 cycles and an annealing and polymerization temperature of 68 °C. The size distribution of PCR- products was analyzed on an 1 % agarose gel (Sambrook et al., supra). Bacterial cultures were mixed with glycerol to a final concentration of 20% and stored at -80 °C.

4. Fluorescence differential display (FDD)

4.1 DNasel digestion

Total RNA (1.) was digested using the MessageClean-Kit (GeneHunter) according to the manufacturers protocol.

4.2 Reverse transcription

Four degenerated primer pools [T7]-Tι₂MA, [T7]-Tι₂MC, [T7]-Tι₂MG and [T7]- T12MT anchoring to poly(A) tails of mRNAs were used, where M is the degenerated position (a mixture of A, C, G). A 17 nt T7 RNA polymerase promoter-derived site (ACGACTCACTATAGGGC) is incorporated which allows the generation of an antisense transcript. For each RNA sample four separate reactions were performed.

200 ng of DNA-free RNA (4.1.) was denatured for 5 min at 70 °C in the presence of 0.2 μM anchor primer [T7]-Tι₂MX and 20 U rRNasin (Promega). After addition of RT buffer (Gibco), 10 mM DTT, 25 μM dNTP and 200 U Superscriptll RTasell (Gibco) on ice, the reaction with a final volume of 20 μl was performed for 5 min at 42 °C and 1 h at 50 °C. The reaction was stopped by heating 15 min at 70 °C.

4.3 PCR

Resulting cDNAs (4.2.) were reamplified in the presence of the same anchor primer labeled with Cy5 and a second primer with 10 nt of arbitrary chosen sequence. A 16 nt segment of the M13 universal reverse (-48) 24mer priming sequence (ACAATTTCACACAGCA) was incorporated in the arbitrary primer [M13r]-ARPXιo for direct sequencing.

1 μl of reverse transcription sample (4.2.) was mixed on ice with 1x PCR buffer (Qiagen), 3.75 mM MgCI₂, 0.35 μM Cy5-[T7]-Tι₂MX, 0.35 μM [M13r]-ARPXιo, 50 μM dNTP and 0.5 U Taq polymerase (Qiagen) in a final volume of 20 μl. PCR was run in a Peltier Thermal Cycler PTC 200 (MJ Research) under the following conditions: 2 min 95 °C, [15 s 92 °C, 30 s 50 °C, 2 min 72 °C]₄, [15 s 92 °C, 30 s 60 °C, 2 min 72 °C]₂₅, 7 min 72 °C, 4 °C. 4.4 Electrop oresis on a 6% deanaturing polyacrylamide gel

The PCR sample (20 μl, 4.3.) was mixed with 6 μl gel loading dye (95% formamide, 20 mM EDTA, 0.005% BPB), denatured for 2 min at 80 °C and separated on a standard sequencing gel (6% polyacrylamide/8.3 M urea) at 55 W for 3 h. The gel was dried on Whatman 3MM paper and fluorescence signals read at 635 nm on a Storm fluorimager (Molecular Dynamics). Data analysis was performed using ImageQuant Software (Molecular Dynamics) as described below (7.3).

4.5 Recovery of PCR fragments from the sequencing gel

Individual bands of interest (4.4.) were cut out of the gel with a scalpel. The gel slice attached to Whatman paper was soaked for 1 h at 37 °C (300 rpm) in 100 μl buffer EB (Qiagen) and incubated at 4 °C over night. Supernatant was purified using the QIAquick PCR purification Kit (Qiagen) as described by the manufacturer. DNA was eluted into 30 μl EB buffer (Qiagen).

4.6 Reamplification of differential display PCR fragments

All PCR fragments recovered from the differential display gel could be reamplified with a set of universal primers, M13r(-48) primer

[AGCGGATAACAATTTCACACAGGA] and T7 primer

[GTAATACGACTCACTATAGGGC]. A 40 μl PCR was set up on ice with 3 μl template (4.5.), 1x PCR buffer, 1.5 mM MgCI₂, 20 μM dNTP, 0.2 μM T7 primer, 0.2 μM M13r(-48) primer and 2 U Taq polymerase (Qiagen) and run as described above (4.3.).

4.7 Electrophoresis on a preparative 1.2% agarose gel

30 μl of reamplified PCR sample were mixed with 6 μl loading dye and separated on an 1.2% agarose/1x TBE gel together with a size standard and a PCR marker (Promega). Bands were cut out with a scalpel and DNA extracted from agarose gel slice using QIAquick gel extraction Kit as described (Qiagen). 1 μl of recovered DNA was used for sequencing.

5. Affymetrix array technology

50 μg total RNA was purified using RNeasy Mini columns (Qiagen) as described by the manufacturer. 5 μg of purified total RNA was used for synthesis of first and second strand cDNA and double stranded cDNA then used to synthesize the biotinylated cRNA probe as recommended by Affymetrix. 15 /g of fragmented labeled cRNA was hybridized to the human genome U95A array and stained by streptavidin-phycoerythrin (SAPE). Signal intensities were amplified using a biotinylated anti-streptavidin antibody and a second SAPE staining step. Data were analyzed by means of Microarray Suite 4.0 (Affymetrix) and Data Mining Tool software provided by Affymetrix. Fold changes in gene expression were analyzed comparing average intensity values of DCM and ICM heart samples with that of normal controls, respectively.

6. Preparation of cDNA libraries and probe synthesis

Since the availability of heart material is very limiting, labeled in vitro transcripts of a cDNA library prepared from heart mRNA were used for dot blot hybridization instead of reverse transcribed mRNA itself.

6.1 Preparation of a cDNA library

5 μg of high quality mRNA (1., 2.) were used to prepare a cDNA library using the cDNA Synthesis Kit and ZAP-cDNA Gigapack III Gold Cloning Kit (Stratagene) as described in the manual with the following modifications:

(a) Packaging and titering: 2.5 μ\ of the ligation reaction were packaged. If the library did not represent at least one million clones, the remaining 2.5 μl were also packaged. After centrifugation of XL1-Blue MRF' culture (50 ml), the cells were gently resuspended in 10 mM MgS0₄ at 4 °C and immediately used for transduction or stored for max 40 h at 4 °C.

(b) Determination of the insert size: 25 plaques were transferred from agar plates used for titering directly into 40 μ\ PCR premix (1x PCR-buffer, 0.25 μM T3 primer, 0.25 μM T7 primer, 200 μM dNTP, 0.085 U Taq DNA- polymerase) and inserts amplified using 35 cycles and an annealing temperature of 48 °C. The insert size was checked on an agarose gel and was in the range of 1-2 kb.

(c) Storage of the library: Libraries were transferred into 50 ml-polypropylene tubes, supplemented with 150 μl 0.3 % chloroform and stored at 4 °C. A part of each library was stored in 7 % DMSO at -80 °C. Mass in v/Vo-excision was done according to the protocol of the ZAP-cDNA Gigapack III Gold Cloning Kit with the following modifications: Transfected XL1 Blue MRF' were grown in 25 ml LB. 5 ml of the supernatant containing single stranded phages was used to infect 20 ml of SOLR cells (Stratagene). Remaining 20 ml of single stranded phages were stored at 4 °C for up to two months. To determine the titer of excised phagemids 10 μl, 1 μl and 0.1 μl of infected SOLR cells were plated on LB/Amp dishes. If the titer was lower than one million, 5 ml or more of the remaining supernatant was used again to infect fresh SOLR cells. Infected SOLR cells (25 ml) were grown in 200 ml LB/Amp over night for plasmid isolation (Plasmid Midi Kit, Qiagen).

6.2 Linearization of the template cDNA library for in vitro transcription

200 μg plasmid DNA were digested with Xhol over night at 37 °C in a volume of 250 μl to linearize the plasmid at the 3' end of the insert. The sample was controlled for complete digestion on an agarose gel, treated with 10 μg/μl Proteinase K (Roche) at 37 °C for 30 min, extracted once with TE saturated phenol (pH 7.5-8) and once with chloroform/isoamylalcohol (24/1) and precipitated in the presence of 0.1 vol 3 M NaOAc (pH 5.2) and 3 volume EtOH. The pellet was washed with 500 μl 75% ethanol, dried at RT for 10 min, dissolved in 150 μl DEPC- treated water and quantified.

1 μg of linearized plasmid was used for an in vitro transcription as described (6.3), omitting the radioactive labeled nucleotide and adding UTP to a final concentration of 10 mM. Following DNasel digestion, the RNA was extracted with phenol/chloroform/isoamylalcohol (24/23/1), precipitated with EtOH and dissolved in 15 μl DEPC-treated water. The yield was in the range of 15-22 μg RNA. 1.5 μl RNA were separated on a formaldehde agarose gel. A smear of transcripts was visible between 0.5 kb and 10 kb with a peak at about 1 kb.

6.3 In vitro transcription

According to the RNA Transcription Kit (Stratagene) 1 μg of linearized template (6.2) was incubated in the presence of 1x transcription buffer, 10 M ATP, 10 mM CTP, 10 mM GTP, 1 mM UTP, 70 μCi [ -³³P]UTP (APB), 0.75 M DTT, 20 U rRNasin. (Promega) and 25 U T3 RNA polymerase for 30 min at 37 °C. After addition of 5 U RNase-free DNasel (Roche) the sample was incubated for 15 min at 37 °C. 25 μl STE-buffer (APB) was added to the probe and the reaction purified using G50 Micro Columns (APB) according to the manufacturers protocol.

6.4 Prehybridization of in vitro transcripts

To suppress probe hybridization to human repetitive DNA, labeled RNA was prehybridized to cot1-DNA. 213 μl DEPC-treated water, 100 μl 20x SSC, 2 μl 20%

SDS and 40 μl cot1-DNA (1 μg/μl, Gibco BRL) were added to 45 μl labeled RNA

(5.3.), denatured at 95 °C for 2 min and incubated for 2 h at 65 °C.

7. Quantitative Dot Blot Analysis

7.1 Transfer of PCR fragments onto nylon membrane

For spotting, approximately 300 ng PCR product (3.2.) or gene-specific control cDNA fragments were mixed with 140 μl 0.4 M NaOH/10 mM EDTA pH 8.0 in 96 well microplates and denatured 10 min at 95 °C. 50 μl of each PCR-fragment (at least 100 ng cDNA) were transferred on a nylon membrane (11.4x7.5 cm, BioRad) using a 384 hole vacuum apparatus (Keutz, custom-made). 50 μl 0.4 M NaOH were added to each position and transferred. The membrane was washed in 2x SSC, dried for at least 1 h at RT and fixed by UV crosslinking (Strataiinker 2400, Stratagene). For each experiment two identical membranes were prepared in parallel.

7.2 Dot blot hybridization and washing

The cDNA filter was soaked in 2x SSC and transferred into a hybridization flask. The membrane was hybridized with 10 ml hybridization solution (6x SSC, 5x Denhardts, 0.2 % SDS, 0.2 % sodium pyrophosphate) supplemented with 50 μg/ml denatured salmon sperm DNA (Typ III, Sigma) at 65 °C for 2 h in an Unitherm 6/12 hybridization oven (UniEquip).

The prehybridization mix was poured off. 200-400 μl of cot 1 -hybridized probe (6.4) were added to 8 ml of hybridization solution (including salmon sperm DNA) preheated to 65 °C. Dot blots were hybridized over night at 65 °C. For washing of cDNA filters all solutions were heated to 65 °C. The membrane was washed twice with 50 ml wash solution 1 (2x SSC, 0.1 % SDS) for 30 min, then twice with 50 ml wash solution 2 (0.1x SSC, 0.1 % SDS) for 30 mjn and wrapped in a keep-fresh foil. The filter was exposed to a phosphor screen for two days and scanned at 450 nm using the Storm Phosphoimager (Molecular Dynamics).

7.3 Data analysis

Signal intensities were calculated using ImageQuant Software (Molecular Dynamics) by subtracting the local background. For comparison of different filters signal intensities were normalized by adjusting the overall intensity of each filter to 100%. In general, two cDNA filters were hybridized successively with 10 probes prepared from different human heart samples.

Dots which represented at least two fold changes in signal intensity comparing the group of DCM heart samples (y) with that of normal controls (x) were selected for further analysis. The probability of type 1 error was calculated to be less than 5% using the Wilcoxon test. This non-parametric statistic algorithm does not assume any distribution of x and y values. If the sample size of one group was smaller than 4 the Wilcoxon test could not be applied. Instead significance of gene regulation was confirmed by a t-test. The t-test assumes that standard deviations of both groups x and y are similar and values distributed according to normal distribution. Independent of the disease individual differences between human samples are expected. They are the result of the different genetic background of individuals, sex, age, environmental and life conditions (e.g. smoking, drinking, nourishment), the status of disease and medical treatment. Especially DCM patients were treated by a number of drugs prior to heart transplantation. It was laid down that the regulation has to be consistent in at least two DCM patients and more or less homogenous in all but one non-failing patient.

Selected clones were grown in 5 ml LB/Amp from glycerol stocks (3.2.). Plasmids were isolated using the Plasmid Mini Kit (Qiagen) and sequenced.

7.4 Stripping of dot blot membranes cDNA filters were transferred into boiling stripping solution (0.1x SSC, 0.5 % SDS) and incubated for 1 h at RT. This procedure was repeated until no more radioactivity could be detected by a Geiger-Mϋller counter. The filter again was wrapped in keep-fresh foil and stored at RT. 8. Full-length cloning:

Full-length cloning was performed using RT-PCR with oligonucleotides priming to the 5'- and 3'- ends of the sequence encoding the open reading frame. PCR- fragments were then purified by agarose gel-electrophoresis followed by gel elution using the gel purification kit from Qiagen. PCR-fragments were finally cloned into P201-DONOR (Life Technologies) or pTOP02.1 (Invitrogen). The cloned cDNAs were verified by sequencing. In addition, in vitro translations were performed using the TNT Quick Coupled Transcription/Translation Systems (Promega) in order to verify the correct molecular weight of the proteins encoded by a given cDNA. The full-length clones were named according to their ID number provided with the suffix "-cds" (xxxxx-cds). The sequence assembled of several ESTs were named according to their ID number provided with suffix "contig" and sequences assembled of known EST and PCR-product were provided with the suffix "cons". The proteins were named according to their ID number provided with the suffix "-pep" (xxxxx-pep).

EXAMPLE 2

EST 40095 (FIG. 1A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control h92 with one from DCM patient h97 (see TABLE 1). The fragment was found to be over- represented in control tissue.

As depicted in FIG. 1 D the identified cDNA fragment is a part of the EST clone AF047439 (FIG. 1 B), which encodes the amino acid sequence AAC39890 (FIG. 1C). The EST clone 40095 is identical to the coding region of a human mRNA of unknown function, which was identified to be expressed in CD34(+) hematopoietic stem and progenitor cells (HSPC001 , Mao et al., 1998). The respective gene is coded on chromosome 1 map P32.

Transcript levels are significantly downregulated by a factor 2.7 in four DCM patients compared to five NF controls (FIG. 1 E). The probability of type 1 error is less than 5% as determined in a t-test and Wilcoxon test. Significant downregulation of 40095 expression in heart tissue of four DCM patients compared to five normal controls indicates that a decreased expression of 40095 is associated with dilated cardiomyopathy. Therefore, the protein is expected to play a causative role in congestive heart failure. The function of the protein is not known so far but since the sequence is highly conserved from mouse to human the protein seems to play an important role.

Upregulation of protein expression by gene therapeutic intervention, compensatory molecules or specific activators seems to be a very promising therapeutic tool to treat heart diseases.

EXAMPLE 3

EST 41237 (FIG. 2 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control h92 with one from DCM patient h97 (see TABLE 1). The fragment was found to be over- represented in the control tissue.

The identified cDNA fragment was found to be a part of the 41237contig ( FIG. 2 B, assembled of AF191652 and AF191653), which encodes the amino acid sequence AAF68859 given in FIG. 2 C (schematic alignment see FIG. 2 D). 41237 is identical to a part of the 3' untranslated region of human diphosphoinositol polyhosphate phosphohydrolase type 2 (NUDT4) of both, alpha (hDIPP2alpha) and beta (hDIPP2beta) isoform distinguished from each other solely by hDIPP2beta possessing one additional amino acid (Caffrey et al., 2000). The enzyme removes the beta-phosphate from all known diphosphoinositol polyphosphates. NUDT4 belongs to the MutT motif protein (or nudix hydrolase) family. Downregulation upon DCM was confirmed by quantitative dot blot analysis. The relative expression level of 41237 is reduced by a factor of 2.2 upon disease (FIG. 2E). The probability of type 1 error is less than 5% as determined in a t-test and Wilcoxon test.

Significant downregulation of 41237 expression in heart tissue of four DCM patients compared to five normal controls indicates that a decreased expression of 41237 is associated with dilated cardiomyopathy. Therefore, the protein is expected to play a causative role in congestive heart failure Diphosphoinositol polyhosphate phosphohydrolase (DIPP) removes the beta- phosphate from all known diphosphoinositol polyphosphates. Diphosphoinositol polyphosphates are members of the inositol-based cell signaling family and comprise a group of highly phosphorylated compounds which have a rapid rate of metabolic turnover through tightly-regulated kinase/phosphohydrolase substrate cycles. Cellular levels are regulated by cAMP and cGMP in a protein kinase- independent manner. These inositides can also sense a specific mode of intracellular Ca²⁺ pool depletion. The phosphohydrolases occur as multiple isoforms, the expression of which is apparently carefully controlled (Safrany et al., 1999). The enzymes are believed to eliminate toxic nucleotide derivatives from the cell and regulate the levels of important signalling nucleotides and their metabolites. Therefore, 41237 can serve as a CHF marker and a specific molecular target for drug development.

EXAMPLE 4

EST 41444 (FIG. 3 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control h92 with one from DCM patient h97 (see TABLE 1). The fragment was found to be over- represented in the control tissue.

The identified cDNA fragment was found to be a part of the EST clone L07956 (FIG. 3 B), which encodes the amino acid sequence AAA58642 (FIG. 3 C; schematic alignment see FIG. 3 D). This amino acid sequence codes for the human 1,4-alpha glucan branching enzyme GBE (Thon et al., 1993). The two major glycogen storage sites are muscle and liver, which provide an energy reservoir for strenous muscular activity. Branching increases the rate of glycogen synthesis and degradation through a higher number of terminal residues. Downregulation upon DCM was confirmed by quantitative dot blot analysis. The relative expression level of 41444 is reduced by a factor of 5 (FIG. 3 E). The probability of type 1 error is less than 1% as determined in a t-test and Wilcoxon test.

Significant downregulation of 41444 expression in heart tissue of four DCM patients compared to five normal controls indicates that a decreased expression of 41444 is associated with dilated cardiomyopathy. Significant repression of glucan branching enzyme will decrease the rate of glycogen synthesis and degradation through a decreased number of terminal residues. Thus, the lowered GBE activity will significantly reduce the glucose reservoir for strenous muscular activity. Therefore, the protein is expected to play a causative role in congestive heart failure.

Type IV glycogenosis (polyglucosan body disease) is a rare congenital autosomal recessive inherited disorder, caused by lack of the branching enzyme (amylo-1 ,4- 1 ,6 transglucosidase). This deficiency leads to storage of abnormal glycogen (polyglucosan bodies) in the liver and other tissues. The clinical onset of the diseases is insidious with non-specific gastrointestinal symptoms followed by progressive hepatic failure. Usually patients die due to hepatic cirrhosis within 4 years. Sometimes myopathy of the heart and skeletal muscle is also present. This corresponds to our results and supports the finding that a decreased expression of 41444 is associated with cardiomyopathies (Nase et. al. 1995). Comparing expression levels of seven tissues the transcript was identified to be expressed at higher levels in the heart (Fig. 3F). Due to its expression predominantly in the heart, GBE can serve as a specific molecular target for drug development.

EXAMPLE 5

EST 41482 (FIG. 4 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control h92 with one from DCM patient h97 (see TABLE 1). The fragment was found to be over- represented in the control tissue.

41482 is identical to the 3' end of the coding sequence of a novel human dermal papilla derived gene (DERP2, Ikeda et al., 2000; cDNA sequence AB009685, FIG. 4 B and included in FIG. 4 F), which codes for the amino acid sequence BAA93049 (FIG. 4 C). Amino acid 1-298 of DERP2 are identical to human PTD010 (cDNA sequence AF078863; amino acid sequence AAD44495), an EST sequence isolated from pituitary tumor (Song et al., 1999). The last 47 amino acids of DERP2 as well as the 3' end of 41482 clearly differ from PTD010. The function of DERP2 is still unknown. A schematic alignment of the different sequences is given in figure 4 D.

Downregulation upon DCM was confirmed by quantitative dot blot analysis. The relative expression level of 41482 is reduced by a factor of 1.7 upon disease (FIG. 4 E). The probability of type 1 error is less than 5% as determined in a t-test and Wilcoxon test.

There is a high probability that the protein is located in the plasma membrane and consists of five membrane-spanning regions (129-145, 161-177, 200-216, 248-264, 275-291). The N-terminus is predicted to be extracellular and the C-terminal side to be cytoplasmic. Two ER membrane retention signals were identified, a XXRR-like motif in the N-terminus (LAAR) and a KKXX-like motif in the C-terminus (GNRK). Significant downregulation of 41482 expression in heart tissue of five DCM patients compared to four normal controls indicates that a decreased expression of 41482 is associated with dilated cardiomyopathy. Therefore, the protein is expected to play a causative role in congestive heart failure. The predicted plasma membrane location indicates a role of 41482 in transport processes or signal transduction. This finding supports the idea that 41482 can be used as a specific molecular target for drug development.

Upregulation of protein expression by gene therapeutic intervention, compensatory molecules or specific activators seems to be a very promising therapeutic tool to treat heart diseases. EXAMPLE 6

EST 52529 (FIG. 5 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control KN2 with one from DCM patient DHZM3 (see TABLE 1). The fragment was found to be over- represented in the control tissue.

The identified cDNA fragment was found to be a part of the coding region and 3' non-coding region of the EST clone NM_006845 (FIG. 5 B), which codes for the amino acid sequence NP_006845 given in FIG. 5 C (schematic alignment see FIG. 5 D), which encodes the human KDEL (Lys-Asp-Glu-Leu) endoplasmatic reticulum protein retention receptor 2 (KDELR2). Retention of luminal endoplasmic reticulum (ER) proteins is mediated via the conserved carboxy-terminal tetrapeptide KDEL that serves as a signal for their retrieval from subsequent compartments of the secretory pathway. The signal is recognized by receptor molecules that are believed to cycle between the Golgi apparatus and the ER. KDELR2 (also referred to as ELP-1) is a novel human homolog of ERD-2 retrieval receptor (Lewis and Pelham, 1992). Both receptors are proposed to return escaped ER resident proteins from the Golgi. Thus, these receptors may provide signals that regulate retrograde traffic between the Golgi and the ER (Hsu et al., 1992). Downregulation upon DCM was confirmed by quantitative dot blot analysis. The relative expression level of 52529 is reduced by a factor of 7.7 upon disease (FIG. 5 E). The probability of type 1 error is less than 5% as determined in a t-test and Wilcoxon test.

Significant downregulation of 52529 expression in heart tissue of six DCM patients compared to five normal controls indicates that a decreased expression of 52529 is associated with dilated cardiomyopathy.

52529 is a novel retrieval receptor proposed to return escaped ER resident proteins from the Golgi and regulate retrograde traffic between the Golgi and the ER. Significant downregulation of 52529 may interfere with the retrieval of KDEL proteins from later stages of the secretory pathway. Thus, minimizing fruitless recycling of secretory proteins and interfering with retrograde transport processes 52529 is expected to play a causative role in congestive heart failure. Upregulation of protein expression by gene therapeutic intervention, compensatory molecules or specific activators seems to be a very promising therapeutic tool to treat heart diseases.

EXAMPLE 7

EST 55253 (FIG. 6 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control KN5 with one from DCM patient 52 (see TABLE 1). The fragment was found to be over- represented in the control tissue.

55253 is identical to part of the coding region and 3' non-coding region of the EST clone U06631 (FIG. 6 B), a human mRNA (H326) which is homologous to murine plasmacytoma expressed PC326 (Bergsagel and Kuehl, 1994). This EST clone codes for the amino acid sequence AAA16607 (FIG. 6 C, schematic alignment see FIG. 6 D).

Downregulation upon DCM was confirmed by quantitative dot blot analysis. The relative expression level of 55253 is reduced by a factor of 1.6 upon disease (FIG. 6 E). The probability of type 1 error is less than 5% as determined in a t-test and Wilcoxon test.

Significant downregulation of 55253 expression in heart tissue of six DCM patients compared to six normal controls indicates that a decreased expression of 55253 is associated with dilated cardiomyopathy. Therefore, the protein is expected to play a causative role in congestive heart failure.

EXAMPLE 8

EST 61024 (FIG. 7 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control KN4 with one from DCM patient h94 (see TABLE 1). The fragment was found to be over- represented in the control tissue. 61024 is identical to a new cDNA clone (AB028963, see FIG. 7 B) of unknown function (KIAA1040, Kikuno et al., 1999), which could be elongated by PCR at the

5^Λend by 292 nt (FIG. 7 C). This EST clone was identified to code for a large protein in the brain with the predicted amino acid sequence 61024pep (FIG. 7 D; schematic alignment see FIG. 7 E). The respective gene is coded on chromosome

12 (AC026115).

Downregulation upon DCM was confirmed by quantitative dot blot analysis. The relative expression level of 61024 is reduced by a factor of 2.1 upon disease (FIG.

7 F). The probability of type 1 error is less than 5% as determined in a t-test and

Wilcoxon test.

Significant downregulation of 61024 expression in heart tissue of five DCM patients compared to five normal controls indicates that a decreased expression of 61024 is associated with dilated cardiomyopathy. Therefore, the protein is expected to play a causative role in congestive heart failure. The function of the protein is not known so far.

EXAMPLE 9

EST 61119 (FIG. 8 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control KN4 with one from DCM patient 94 (see TABLE 1). The fragment was found to be over- represented in the control tissue.

61119 is identical to a part of the EST clone AF115509 (FIG. 8 B), the coding region of leucine rich repeat of fligthless I (LRR FLI-I) interacting protein 2 (LRRFIP2) encoded by the amino acid sequence AAA16607 (FIG. 8 C, schematic alignment see FIG. 8 D). This protein was identified by yeast two-hybrid to interact via its conserved coiled-coil domain with leucine-rich repeat (LRR) domain of human fligthless I (Fong and de Couet, 1999). LRRFIP2 is a novel gene that shares sequence homology with LRRFIP1 and FLAP-1. LRRFIP1 and LRRFIP2 both express alternative splice variants in heart and skeletal muscle tissue. Both genes are related and seem to arose from gene duplication. Downregulation upon DCM was confirmed by quantitative dot blot analysis. The relative expression level of 61119 is reduced by a factor of 2 upon disease (FIG. 8 E). The probability of type 1 error is less than 5% as determined in a t-test and Wilcoxon test.

Significant downregulation of 61119 expression in heart tissue of five DCM patients compared to five normal controls indicates that a decreased expression of 61119 is associated with dilated cardiomyopathy.

Flightless-I (flil) is a novel member of the gelsolin family that is important for actin organization during Drosophila embryogenesis and myogenesis (Liu and Yin, 1998). Drosophila flil and the human homolog FLI both contain the classic gelsolin 6-fold segmental repeats and an amino-terminal extension of 16 tandem leucine- rich repeats (LRR). Although the association between actin and the gelsolin-like domain of human FLI has been established, its biological role is unknown. The LRR repeat forms an amphipathic beta-alpha structural unit that mediates protein- protein interaction between FLI and the coiled-coil structure of LRRFIP2. Since FLI is involved in structural reorganization in heart and skeletal muscle and LRRFIP2 directly interacts with FLI LRRFIP2 is expected to play a causative role in congestive heart failure. Moreover, the LRR domain is highly homologous to those of three proteins involved in Ras-mediated signaling: S. cerevisiae adenylyl cyclase, C. elegans SUR-8, and mammalian RSP-1 (Goshima et al., 1999). FLI-1 may be involved in regulation of the actin cytoskeleton through Ras. This supports the idea that 61119 plays a central role in muscle degeneration throughout disease and therefore can be used as a specific molecular target for drug development. Upregulation of protein expression by gene therapeutic intervention, compensatory molecules or specific activators seems to be a very promising therapeutic tool to treat heart diseases. EXAMPLE 10

EST 62139 (FIG. 9 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control KN4 with one from DCM patient h94 (see TABLE 1). The fragment was found to be over- represented in the control tissue.

62139 was found to be a part of 62139contig (FIG. 9 B), assembled of X97324 (and further fragments), which codes for the amino acid sequence CAA65989 (FIG. 9 C; schematic alignment see FIG. 9 D). This amino acid sequence encodes the human adipophilin (ADFP), a specific marker for adipocyte cell differentiation and lipid accumulation in a variety of cells. Most mammalian cells package neutral lipids into droplets that are surrounded by a monolayer of phospholipids and a specific set of proteins including adipophilin, which is found in a wide array of cell types. The 50-kD membrane-associated protein is associated with the surface of lipid droplets and was revealed as a possible new marker for the identification of specialized differentiated cells containing lipid droplets and for diseases associated with fat-accumulating cells (Heid et al., 1998).

Transcript levels are significantly downregulated by a factor 2.1 in five DCM patients compared to five normal controls (FIG. 9 E). The probability of type 1 error is less than 5% as determined in a t-test and Wilcoxon test. A downregulation of 62139 upon DCM has also been found using the Affymetrix array technology. Transcript levels^'are significantly downregulated by a factor 1.91 in 11 DCM patients compared to ten normal controls (FIG. 9 F). The probability of type 1 error for DCM is 5.8% and 3.5% as determined in a t-test and Mann- Whitney test. In addition, transcript levels are downregulated by a factor of 1.55 in 9 ICM patients compared to ten normal controls (FIG. 9 F). The probability of type 1 is 13.2% and 68.7% as determined in a t-test and Mann-Whitney test, respectively. The comparison of the mean values of DCM patients, ICM patients and normal controls is shown in FIG. 9 G.

Significant downregulation of 62139 expression in heart tissue of five DCM patients compared to the same number of normal controls indicates that a lowered expression of 62139 is associated with dilated cardiomyopathy. ADFP is a specific marker of lipid accumulation in diverse cell types and diseases. In cardiomyopathy the diseased heart uses fatty acids as main energy source instead of glucose (Paolisso et al., 1994, Lommi et al., 1998). A lowered expression of 62139 by a factor of 2 upon DCM therefore may directly correspond to a decreased amount of lipids stored in lipid droplets. Therefore 62139 can be used as a specific marker for CHF and may also be a molecular target for drug development. Upregulation of protein expression by gene therapeutic intervention, compensatory molecules or specific activators seems to be a very promising therapeutic tool to treat heart diseases.

EXAMPLE 11

EST 62140 (FIG. 10 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control KN4 with one from DCM patient h94 (see TABLE 1). The fragment was found to be over- represented in the control tissue.

The identified cDNA fragment was found to be a part of the 62140contig (FIG. 10 B), which was assembled of AW952950 and AI653533 beside/and other ESTs (schematic alignment see FIG. 10 C). For the 62140contig sequence of 1373 nt as well as for the homologous EST sequences annotated in Genbank no clear open reading frame could be identified.

Downregulation upon DCM was confirmed by quantitative dot blot analysis. The relative expression level of 62140 is reduced by a factor of 2.1 upon disease (FIG. 10 D). The probability of type 1 error is less than 5% as determined in a t-test and Wilcoxon test.

Significant downregulation of 62140 expression in heart tissue of five DCM patients compared to five normal controls indicates that a decreased expression of 62140 is associated with dilated cardiomyopathy. Therefore, the unknown protein is expected to play a causative role in congestive heart failure. Moreover, statistical analysis of expressed ESTs according to LabOnWeb (Compugen) revealed that the protein is predominantly expressed in the heart. This finding supports the idea that 66214 is a specific molecular target for drug development. Upregulation of protein expression by gene therapeutic intervention, compensatory molecules or specific activators seems to be a very promising therapeutic tool to treat heart diseases.

Abbreviate >ns

aa amino acids

ACEI inhibitors of angiotensin converting enzyme

Amp ampicillin

BPB bromphenol blue cDNA copy deoxyribonucleic acid

CHF congestive heart failure

DCM dilative cardiomyopathy ddH₂0 double-distilled water

DEPC diethyl pyrocarbpnate

EtOH ethanol f female

FDD fluorescence differential display

GAPDH glyceraldeyde-3-phosphate dehydrogenase

HCM hypertrophic cardiomyopathy

ICM ischemic cardiomyopathy

ID identity m male mRNA messenger ribonucleic acid

NaOAc sodium acetate

NF non-failing/healthy patient

RT room temperature

SSH suppression subtractive hybridization

U units vol volume

XC xylene cyanol References

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Biol Chem 273, 7920-7927. Lommi J, Kupari M, Yki-Jarvinen H (1998) Free fatty acid kinetic oxidation in congestive heart failure. Am J Caldiol. 81 (1), 45-50. Loughney K, Martins TJ, Harris EA, Sadhu K, Hicks JB, Sonnenburg WK, Beavo

JA and Ferguson K (1996) Isolation and characterization of cDNAs corresponding to two human calcium, calmodulin-regulated, 3',5'-cyclic nucleotide phosphodiesterases. J Biol Chem 271 , 796-806. Mao M, Fu G, Wu JS, Zhang QH, Zhou J, Kan LX, Huang QH, He KL, Gu BW, Han

ZG, Shen Y, Gu J, Yu YP, Xu SH, Wang YX, Chen SJ and Chen Z (1998)

Identification of genes expressed in human CD34(+) hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning. Proc NatlAcad Sci U S A 95, 8175-8180. Nase S, Kunze KP, Sigmund M, Schroeder JM, Shin Y, Hanrath P (1995) A new variant of type IV glycogenosis with primary cardiac manifestation and complete branching enzyme deficiency. In vivo detection by heart muscle biopsy. Eur

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Fujimoto E and Kataoka T (1999) Characterization of a novel Ras-binding protein Ce-FLI-1 comprising leucine-rich repeats and gelsolin-like domains.

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P, Varricchio M, D'Onofio F (1994) Total-body myocardial substrate oxidation in congestive heart failire. Metabolism 42(2), 174-9. Rahmani, Z and Siddiqui, A (1998) Human voltage dependent anion channel form

3 (Unpublished). Rebbe NF, Ware J, Bertina RM, Modrich P and Stafford DW (1987) Nucleotide sequence of a cDNA for a member of the human 90-kDa heat-shock protein family. Gene 53, 235-245. Rostovtseva T and Colombini M (1997) VDAC channels mediate and gate the flow of ATP: implications for the regulation of mitochondrial function. Biophys J 72,

1954-1962. Safrany ST, Caffrey JJ, Yang X, Shears SB (1999) Diphosphoinositol polyphosphates: the final frontier for inositide research. Biol Chem 380, 945-951 Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory

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7513

Claims

A method for identifying a subject at risk for a disease of the heart, comprising the step of quantitating the amount of at least one RNA in the heart tissue of the subject, whereby

(a) said at least one RNA encodes an amino acid sequence selected from the group consisting of:

(aa) the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10;

(ab) an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to the amino acid sequence of (aa);

(ac) the amino acid sequence of (aa) with at least one conservative amino acid substitution;

(ad) an amino acid sequence that is an isoform of the amino acid sequence of any of (aa) to (ac); and

(ae) an^" amino acid that is encoded by a DNA molecule the complementary strand of which hybridizes in 4xSSC, 0.1% SDS at 65°C to the DNA molecule encoding the amino acid sequence of (aa), (ac) or (ad); and/or

(b) said at least one RNA is transcribed from the DNA sequence of SEQ ID NO: 11 , the DNA sequence of SEQ ID NO: 12, the DNA sequence of SEQ ID NO: 13, the DNA sequence of SEQ ID NO: 14, the DNA sequence of SEQ ID NO: 15, the DNA sequence of SEQ ID NO: 16, the DNA sequence of SEQ ID NO: 17, the DNA sequence of SEQ ID NO: 18, the DNA sequence of SEQ ID NO: 19, the DNA sequence of SEQ ID NO: 20, the DNA sequence of SEQ ID NO: 21 , the DNA sequence of SEQ ID NO: 22, the DNA sequence of SEQ ID NO: 23, the DNA sequence of SEQ ID NO: 24, the DNA sequence of SEQ ID NO: 25, the DNA sequence of SEQ ID NO: 26, the DNA sequence of SEQ ID NO: 27 or the DNA sequence of SEQ ID NO: 28 or a degenerate variant thereof.

The method according to claim 1 , wherein the amount of the said RNA is quantitated using a nucleic acid probe which is a nucleic acid comprising a sequence selected from the group consisting of:

(a) the DNA sequence of SEQ ID NO: 11 , the DNA sequence of SEQ ID NO: 12, the DNA sequence of SEQ ID NO: 13, the DNA sequence of SEQ ID NO: 14, the DNA sequence of SEQ ID NO: 15, the DNA sequence of SEQ ID NO: 16, the DNA sequence of SEQ ID NO: 17, the DNA sequence of SEQ ID NO: 18, the DNA sequence of SEQ ID NO: 19, the DNA sequence of SEQ ID NO: 20, the DNA sequence of SEQ ID NO: 21, the DNA sequence of SEQ ID NO: 22, the DNA sequence of SEQ ID NO: 23, the DNA sequence of SEQ ID NO: 24, the DNA sequence of SEQ ID NO: 25, the DNA sequence of SEQ ID NO: 26, the DNA sequence of SEQ ID NO: 27 or the DNA sequence of SEQ ID NO: 28 or a degenerate variant thereof;

(b) a DNA sequence at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to the DNA sequence of (a);

(c) a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; each of said amino acid sequences having at least one conservative amino acid substitution; (d) a nucleic acid sequence that encodes an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to the amino acid sequence of

(c);

(g) a fragment of at least 15 nucleotides in length of (a) to (f); and

(h) a nucleic acid probe comprising a sequence that specifically hybridizes under physiological conditions to the nucleotide sequence selected from the group consisting of:

(i) the DNA sequence of the RNA transcribed from the DNA sequence of SEQ ID NO: 11 , the DNA sequence of SEQ ID NO: 12, the DNA sequence of SEQ ID NO: 13, the DNA sequence of SEQ ID NO: 14, the DNA sequence of SEQ ID NO: 15, the DNA sequence of SEQ ID NO: 16, the DNA sequence of SEQ ID NO: 17, the DNA sequence of SEQ ID NO: 18, the DNA sequence of SEQ ID NO: 19, the DNA sequence of SEQ ID NO: 20, the DNA sequence of SEQ ID NO: 21 or the DNA sequence of SEQ ID NO: 22, the DNA sequence of SEQ ID NO: 23, the DNA sequence of SEQ ID NO: 24, the DNA sequence of SEQ ID NO: 25, the DNA sequence of SEQ ID NO: 26, the DNA sequence of SEQ ID NO: 27 or the DNA sequence of SEQ ID NO: 28;

(ii) a DNA sequence at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to the DNA sequence of (i);

(iii) a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10 with at least one conservative amino acid substitution;

(iv) a nucleic acid sequence that encodes an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to the amino acid sequence of (iii);

(v) a nucleic acid sequence that encodes the amino acid sequence of (iii) with at least one conservative amino acid substitution;

(vi) a nucleic acid sequence that hybridizes in 2xSSC, 0.1% SDS at 65°C to the DNA molecule encoding the amino acid sequence of (iii), (iv) or (v); and

(vii) a fragment of at least 15 nucleotides in length of (i) to (vi).

3. A method for identifying a subject at risk for a disease of the heart, comprising the step of quantitating the amount of a polypeptide selected from the group consisting of:

(a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10;

(b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99%o identical to the amino acid sequence of (a); and

(c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, in the heart tissue or the serum of the blood of the subject.

4. The method according to claim 3, wherein the amount of the said polypeptide is quantitated using an antibody or an antigen-binding portion of said antibody that specifically binds a polypeptide selected from the group consisting of:

(b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to the amino acid sequence of (a); and

(c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution.

5. The method according to claim 4, wherein said antibody or antibody binding portion is or is derived from a human antibody or a humanized antibody.

6. The method according to claim 4 or claim 5, wherein the antibody, the binding portion or derivative thereof is detectably labeled.

7. The method of claim 6, wherein said derivative of said antibody is an scFv fragment.

8. The method of claim 1 or 2, wherein said RNA is obtained from heart tissue.

9. The method of any one of claims 3 to 7 wherein said polypeptide is quantitated in heart tissue.

10. The method of any one of claims 1 , 2 and 8 further comprising the step of normalizing the amount of RNA against a corresponding RNA from a healthy subject or cells derived from a healthy subject.

11. The method of any one of claims 3 to 7 and 9 further comprising the step of normalizing the amount of polypeptide against a corresponding polypeptide from a healthy subject or cells derived from a healthy subject.

12. A method for identifying a compound that increases the level in heart tissue of a polypeptide selected from the group consisting of:

(b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99 identical to the amino acid sequence of (a); and

(c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, said method comprising the steps of:

(1) contacting a DNA encoding said polypeptide under conditions that would permit the translation of said polypeptide with a test compound; and

(2) detecting an increased level of the polypeptide relative to the level of translation obtained in the absence of the test compound.

13. A method for identifying a compound that specifically binds to a polypeptide having an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; said method comprising the steps of

(1) providing said polypeptide; and

(2) identifying a compound that is capable of binding said polypeptide.

14. The method of claim 13, wherein said binding results in activation of said polypeptide.

15. A monoclonal antibody or derivative thereof that specifically binds to a polypeptide having an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10.

16. A method for identifying a compound that increases the level in heart tissue of an mRNA encoding a polypeptide selected from the group consisting of:

(b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, said method comprising the steps of

(1) contacting a DNA giving rise to said mRNA under conditions that would permit transcription of said mRNA with a test compound; and

(2) detecting an increased level of the mRNA relative to the level of transcription obtained in the absence of the test compound.

17. A transgenic non-human mammal whose somatic and germ cells comprise at least one gene encoding a functional polypeptide selected from the group consisting of.

(c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, said functional polypeptide has been modified, said modification being sufficient to decrease the amount of said functional polypeptide expressed in the heart tissue of said transgenic non-human mammal, wherein said transgenic non-human mammal exhibits a disease of the heart.

18. The transgenic non-human mammal according to claim 17, wherein said disrupted gene was introduced into the non-human mammal or an ancestor thereof, at an embryonic stage.

19. A transgenic non-human mammal according to claim 17 or 18, wherein the modification is inactivation or suppression of said gene(s) or leads to the reduction of the synthesis of the corresponding protein(s).

20. A method for identifying a compound that increases the expression of a polypeptide in heart tissue, the polypeptide being selected from the group consisting of:

(1) contacting a transgenic non-human mammal according to any one of claims 15 to 17 with a test compound, and

(2) detecting an increased level of expression of said polypeptide relative to the expression in the absence of said test compound.

21. The method according to claim 20, wherein the test compound prevents or ameliorates a disease of the heart in said transgenic non-human mammal.

22. A method for identifying one or a pluratiy of isogenes of a gene coding for a polypeptide selected from the group consisting of: the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; said method comprising the steps of

(1) providing nucleic acid coding for said polypeptide or a part thereof; and

(2) identifying a second nucleic acid that (i) has a homology of 60%, preferably at least 80%, especially at least 90%, advantageously at least 99 or (ii) hybridizes in 4xSSC, 0.1 SDS at 45°C to the nucleic acid molecule encoding said amino acid sequences.

23. A method for identifying one or a plurality of genes whose expression in heart tissue is modulated by inhibiting or decreasing the expression of a polypeptide selected from the group consisting of:

(c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, or of an mRNA encoding said polypeptide, said modulation being indicative of a disease of the heart, said method comprising the steps of: (1) contacting a plurality of heart tissue cells with a compound that inhibits or decreases the expression of said polypeptide under conditions that permit the expression of said polypeptide in the absence of a test compound, and (2) comparing a gene expression profile of said heart cell in the presence and in the absence of said compound.

24. A method for identifying one or a plurality of genes whose expression in heart tissue is modulated by the inhibition or decrease of the expression of a polypeptide selected from the group consisting of:

(c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, or of an mRNA encoding said polypeptide, said modulation being indicative of a disease of the heart, said method comprising the steps of.

(1) providing expression profiles of

(i) a plurality of heart tissue cells from or derived from a heart of a subject suffering from a disease of the heart; and

(ii) a plurality of heart tissue cells from or derived from a subject not suffering from a disease of the heart; and

(2) comparing the expression profiles (i) and (ii).

25. The method of claim 23 further comprising the steps of

(3) determining at least one gene that is expressed at a lower or higher level in the presence of said compound; and (4) identifying a further compound that is capable of raising or lowering the expression level of said at least one gene.

26. The method of claim 24 further comprising the steps of

(3) determining at least one gene that is expressed at a lower or higher level in said heart tissue cells from or derived from a heart of a subject suffering from a disease of the heart; and

(4) identifying a further compound that is capable of raising or lowering the expression level of said at least one gene.

27. A method for identifying a protein or a plurality of proteins in heart tissue whose activity is modulated by a polypeptide having the amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; said method comprising the steps of

(1) providing said polypeptide; and

(2) identifying a further protein that is capable of interacting with said polypeptide.

28. The method of any one of claims 12 to 14, 16, 20, 21 , 23, 25, and 26, wherein said compound is a small molecule or a peptide derived from an at least partially randomized peptide library.

29. A method of refining a compound identified by the method of any one of 12 to 14, 16, 20, 21, 23, 25, 26 or 28, said method comprising the steps of

(1) identification of the binding sites of the compound and the DNA or mRNA molecule by site-directed mutagenesis or chimeric protein studies;

(2) molecular modeling of both the binding site of the compound and the binding site of the DNA or mRNA molecule; and

(3) modification of the compound to improve its binding specificity for the DNA or mRNA.

30. The method of any one of claims 12 to 14, 16, 20, 21, 23, 25, 26, 28 and 29, wherein said compound is further refined by peptidomimetics.

31. A method of modifying a compound identified or refined by any one of claims 12 to 14, 16, 20, 21 , 23, 25, 26, 28 to 30 as a lead compound to achieve

(i) modified site of action, spectrum of activity, organ specificity, and/or

(ii) improved potency, and/or

(iii) decreased toxicity (improved therapeutic index), and/or

(iv) decreased side effects, and/or

(v) modified onset of therapeutic action, duration of effect, and/or

(vi) modified pharmakinetic parameters (resorption, distribution, metabolism and excretion), and/or

(vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or

(viii) improved general specificity, organ/tissue specificity, and/or

(ix) optimized application form and route by

(i) esterification of carboxyl groups, or

(ii) esterification of hydroxyl groups with carbon acids, or

(iii) esterification of hydroxyl groups to, e.g. phosphates, pyrophosphates or sulfates or hemi succinates, or

(iv) formation of pharmaceutically acceptable salts, or

(v) formation of pharmaceutically acceptable complexes, or

(vi) synthesis of pharmacologically active polymers, or

(vii) introduction of hydrophylic moieties, or (viii) introduction/exchange of substituents on aromates or side chains, change of substituent pattern, or (ix) modification by introduction of isosteric or bioisosteric moieties, or (x) synthesis of homologous compounds, or (xi) introduction of branched side chains, or (xii) conversion of alkyi substituents to cyclic analogues, or (xiii) derivatisation of hydroxyl group to ketales, acetales, or (xiv) N-acetylation to amides, phenylcarbamates, or (xv) synthesis of Mannich bases, imines, or (xvi) transformation of ketones or aldehydes to Schiffs bases, oximes, acetales, ketales, enolesters, oxazolidines, thiozolidines or combinations thereof.

32. A method for inducing a disease of the heart in a non-human mammal, comprising the step of contacting the heart tissue of said mammal with a compound that inhibits or decreases the expression of a polypeptide selected from the group consisting of:

33. The method according to claim 32, wherein said compound that inhibits or decreases is a small molecule, an antibody or an aptamer that specifically binds said polypeptide.

34. A method of producing a pharmaceutical composition comprising formulating the compound identified, refined or modified by the method of any of the preceding claims with a pharmaceutically active carrier or diluent.

35. A method for preventing or treating a disease of the heart in a subject in need of such treatment, comprising the step of increasing the level of a polypeptide in the heart tissue of a subject, said polypeptide being selected from the group consisting of:

(c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, in the heart tissue of the subject.

36. A method of preventing or treating a disease of the heart in a subject in need of such treatment comprising the step of increasing the level of mRNA encoding a polypeptide in the heart tissue of a subject, said polypeptide being selected from the group consisting of:

(c) a polypeptide having the amino acid sequence of (a) with at least oneconservative amino acid substitution.

37. The method of claims 35 or 36, wherein such increase is effected by administering the pharmaceutical composition obtained by the method of claim 31.

38. The method of claim 35 or 36, wherein such an increase is effected by introducing the nucleic acid sequence recited in claim 2 into the germ line or into somatic cells of a subject in need thereof.

39. The method of any of the preceding claims, wherein said disease of the heart is congestive heart failure, dilative cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, specific heart muscle disease, rhythm and conduction disorders, syncope and sudden death, coronary heart disease, systemic arterial hypertension, pulmonary hypertension and pulmonary heart disease, valvular heart disease, congenital heart disease, pericardial disease or endocarditis.

40. A method for identifying subjects at risk for congestive heart failure comprising the step of detecting decreased diphosphoinositol polyphosphate phosphodihydrolase type II beta (DIPP2β) activity in the heart tissue of a subject.

41. A method for identifying a subject at risk for congestive heart failure, said method comprising detecting increased levels of β-phosphorylated diphosphoinositol polyphosphates.

42. A method for preventing or treating congestive heart failure in a subject, said method comprising the step of contacting the heart tissue of said subject with a compound that increases the expression and/or the activity of DIPP2β

43. A method for preventing or treating congestive heart failure in a subject, said method comprising the step of increasing the removal of β-phosphate from diphosphoinositol polyphosphates in the heart tissue of said subject.

44. The method according to claim 43, wherein the expression of DIPP2β is increased in said heart tissue.

45. The method according to claim 43, wherein the expression of DIPP2α is increased in said heart tissue.

46. The method according to claim 44 or 45, where the expression of an endogenous gene is increased in said heart tissue.

47. The method according to claim 44 or 45, where the expression of an exogenous gene is increased in said heart tissue.

48. The method according to claim 43, wherein the activity of DIPP2β is increased in said heart tissue.

49. The method according to claim 43, wherein the activity of DIPP2α is increased in said heart tissue.

50. A method for preventing or treating congestive heart failure in a subject comprising the step of decreasing the amount of β-phosphorylated diphosphoinositol polyphosphates in the heart tissue of said subject.

51. A method for identifying a compound for preventing or treating congestive heart failure, said method comprising the steps of:

(a) contacting DIPP2β or DIPP2α with a substrate for DIPP2β or DIPP2α and a test compound, and

(b) determining whether the removal of β-phosphate from the substrate is increased in the presence of the test compound.

52. A method for identifying a compound for preventing or treating congestive heart failure, said method comprising the steps of:

(a) contacting a heart tissue cell expressing DIPP2β or DIPP2α with a test compound; and

(b) determining whether removal of β-phosphate from diphosphoinositol polyphosphates is increased.

53. A method for preventing or treating congestive heart failure in a subject comprising the step of increasing the glucose level in heart tissue in said subject.

54. The method according to claim 53, wherein said glucose level is increased by administering glucose to said subject.

55. The method according to claim 53, wherein said glucose level is increased by increasing the amount of glycogen storage in said heart tissue.

56. The method according to claim 55, wherein glycogen storage is increased by increasing the expression and/or activity of a glucan branching enzyme (GBE).

57. The method according to claim 56, wherein the GBE is 1 ,4- -GBE.

58. A method for preventing or treating congestive heart failure in a subject, comprising increasing the activity of DERP2.

59. A method for preventing or treating congestive heart failure in a subject, comprising increasing the DERP2-mediated signal transduction in the heart tissue of said subject.

60. A method for preventing or treating congestive heart failure in a subject, comprising increasing DERP2-mediated cellular transport in the heart tissue of said subject.

61. The method according to any one of claims 58 to 60, comprising the step of increasing the expression of DERP2 on the plasma membrane of cells in said heart tissue.

62. The method according to any one of claims 58 to 60, comprising the step of contacting said heart tissue with a compound that is a DERP2 activator.

63. Use of a compound of any one of the claims 12 to 14, 16, 20, 21 , 23, 25, 26, 28, 41 , 46, a refined or modified compound of any one of the claims 29, 30 or 31 , or a monoclonal antibody of the claim 15 for the manufacture of a pharmaceutical composition for the prophylaxis or treatment of heart diseases, especially congestive heart failure.